Alpha-fetoprotein antagonizes X-linked inhibitor of apoptosis protein anticaspase activity and disrupts XIAP–caspase interaction Elena Dudich1,2, Lidia Semenkova1,2, Igor Dudich1,2, Alexander Denesyuk3, Edward Tatulov2 and Timo Korpela4 Institute of Immunological Engineering, Lyubuchany, Russia JSC BioSistema, Moscow, Russia ˚ Department of Biochemistry and Pharmacy, Abo Akademi University, Turku, Finland Joint Biotechnology Laboratory, Turku University, Finland Keywords apoptosis; apoptosome; caspases; a-fetoprotein; X-linked inhibitor of apoptosis protein Correspondence E Dudich, Institute of Immunological Engineering, 142380, Lyubuchany, Moscow Region, Chekhov District, Russia Fax ⁄ Tel: +7 095 996 1555 E-mail: elena_dudich@mail.ru (Received 28 February 2006, revised May 2006, accepted 22 June 2006) doi:10.1111/j.1742-4658.2006.05391.x Previous results have shown that the human oncoembryonic protein a-fetoprotein (AFP) induces dose-dependent targeting apoptosis in tumor cells, accompanied by cytochrome c release and caspase activation AFP positively regulates cytochrome c ⁄ dATP-mediated apoptosome complex formation in a cell-free system, stimulates release of the active caspases and and displaces cIAP-2 from the apoptosome and from its complex with recombinant caspases and [Semenkova et al (2003) Eur J Biochem 270, 276–282] We suggested that AFP might affect the X-linked inhibitor of apoptosis protein (XIAP)–caspase interaction by blocking binding and activating the apoptotic machinery via abrogation of inhibitory signaling We show here that AFP cancels XIAP-mediated inhibition of endogenous active caspases in cytosolic lysates of tumor cells, as well as XIAP-induced blockage of active recombinant caspase in a reconstituted cell-free system A direct protein–protein interaction assay showed that AFP physically interacts with XIAP molecule, abolishes XIAP–caspase binding and rescues caspase from inhibition The data suggest that AFP is directly involved in targeting positive regulation of the apoptotic pathway dysfunction in cancer cells inhibiting the apoptosis protein function inhibitor, leading to triggering of apoptosis machinery Apoptotic dysfunction plays a key role in cancer progression and leads to chemotherapeutic and radiotherapeutic resistance [1–3] Many cancer therapeutic agents operate by inducing apoptosis and are ineffective in conditions of impaired apoptosis signaling Novel strategies for cancer therapy are aimed at discovering molecular targets involved in the induction of apoptosis in normal and tumor cells, and at selectively regenerating the apoptosis propensity in cancer cells Apoptosis is induced by two different mechanisms: the extrinsic or receptor-dependent pathway and the intrinsic or mitochondria-dependent pathway [4] Triggering of either pathway results in the initiation of caspase cascade activation events Caspases are generally divided into two groups according to their functional hierarchy and substrate specificity The initiator caspase family includes caspases 2, 8, 9, 10 and 12, and is characterized by the presence of N-terminal prodomains DED or CARD, which are involved in Abbreviations Ac-DEVD-AMC, Ac-Asp-Glu-Val-Asp-7-amino-4-methyl coumarin; AFP, a-fetoprotein; IAP, inhibitor of apoptosis protein; IBM, IAP-binding motif; RFU, relative fluorescence units; XIAP, X-linked inhibitor of apoptosis protein FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS 3837 AFP antagonizes XIAP function E Dudich et al interactions with certain adapter molecules to form death-inducing signaling complex or apoptosome [3–5] Effector caspases 3, and exist in the cytosol as inactive zymogens and are activated via a proteolytic cascade started by the initiator caspases [5] Molecular pathways leading to apoptosis are evolutionarily conserved and are regulated by specific cellular proteins Some, such as Bcl-2, control the release of proapoptotic proteins from the mitochondria [6] Others, including various cellular inhibitor-of-apoptosis proteins (IAPs), bind directly to active caspases and act as natural inhibitors of caspase activity [7] The IAP family, currently identified in humans consists of X-linked inhibitor of apoptosis protein (XIAP), ILP-2, cIAP1, cIAP2, ML-IAP, NAIP, survivin, and livin [7–9] All IAPs have so-called conservative BIR domains, which are responsible for their interaction with caspases XIAP is the most potent of all known IAPs and contains three BIR domains The third BIR domain (BIR3) selectively targets caspase 9, whereas BIR2 and the linker region between BIR1 and BIR2 inhibit effector caspases and [5,10] This inhibition can be relieved by IAP antagonists, which bind to IAPs preventing caspase binding [5,8,9,11–13] Recent studies have revealed that binding of IAP antagonists to IAPs may stimulate their auto-ubiquitination and degradation, thereby preventing caspase inhibition [14,15] Recognition of XIAP as a direct inhibitor of caspases makes it an attractive therapeutic target This led to an active search for any suitable molecular inhibitor capable of easily penetrating a tumor cell to block XIAP activity in the cytosol [8,9,16] The discovery of endogenous regulators of IAP activity enhanced these investigations Several intracellular inhibitory IAPs have been characterized in humans, namely, Smac ⁄ DIABLO, Omi ⁄ HtrA2, GSPT1 ⁄ eRF3, ARTS, and XAF1 [17–21] However, only the first and best characterized anti-IAP Smac ⁄ DIABLO is currently known to be directly involved in the regulation of apoptosis Other anti-IAPs, such as Omi ⁄ HtrA2 or GSPT1 ⁄ eRF3, seem to have a primary physiological role that is not directly related to XIAP ⁄ caspase regulation [8,18,19] Smac ⁄ DIABLO is released from mitochondria into the cytosol during apoptosis, wherein it can bind to XIAP [17] The main highly conserved functional motif common to all IAP antagonists, was termed the IAP-binding motif (IBM), and became a target for finding novel potential inhibitors of IAP [8,9,16] The motif ATPF ⁄ AVPI was first characterized in caspase and Smac ⁄ DIABLO was characterized as being responsible for binding to BIR3 of XIAP [22] Smac-derived peptides modeling their XIAP-binding site, bind to recombinant BIR3 domains in vitro 3838 [18–23] Recent studies have set out to design small molecular drugs carrying the IBMs [5,11–13] or artificial chimerical peptides composed of the IBM sequence fused to a carrier peptide [23] The cell-permeable Smac peptides allowed the apoptosis resistance and chemoresistance of cancer cells with a high level of XIAP to be overcome in vitro and in vivo, as documented [13,23] Despite the strong molecular basis for interaction with XIAP, natural Smac-derived peptides and other artificial IBM-based chimeric constructions have several intrinsic limitations (e.g poor in vivo stability and very low bioavailability) making them unsuitable for the treatment of cancer [8,9,23] The other known natural XIAP-binding proteins cannot act as anticancer drugs because of their exclusive intracellular location Therefore, the search for other XIAP-interacting and cell-membrane-penetrating drugs is a highly desirable goal Recently, it was discovered that the well-known oncofetal antigen a-fetoprotein (AFP) is able to induce apoptosis selectively in tumor cells without any toxicity towards normal cells and tissues [24–28] AFP is one of the major serum embryonic proteins involved in the regulation of growth and the development of immature embryonic tissues [29–31] The specific expression and internalization of AFP is restricted to developing cells, such as embryonic cells, activated immune cells and tumor cells, which suggests that it has an important regulatory role in cell growth and differentiation [32– 35] AFP expression is blocked completely after birth and is recovered only after malignant transformation [29–31] Various researchers have documented the existence of specific receptor-dependent mechanisms responsible for the active endocytosis of AFP by malignant cells [34–36] AFP has been well characterized as a transport protein delivering natural ligands such as fatty acids, hormones, and heavy metals to developing cells [29] The specific expression and internalization of AFP by developing cells, such as embryonic cells or tumor cells, together with the properties of the transport protein make AFP very attractive for tumor-targeting therapy [29,30,33] The growth-regulatory activity of AFP and AFP derivatives has been demonstrated by various authors [24–28,37–43] Special interest has focused on the tumor-suppressive effects of AFP and its peptide derivatives [24–28,38, 39,42] The growth-suppressive activity of AFP can be realized by inducing apoptosis in many types of tumor or activated immune cells [24–28,41] AFP can trigger apoptosis in tumor cells via activation of caspase 3, independent of the membrane-receptor signaling [26] AFP stimulates formation of the apoptosome complex, and enhances recruitment and activation of caspases FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS E Dudich et al and by displacing cIAP-2 from the apoptosome and from its complex with recombinant caspases and [28] Based on the molecular mechanisms of AFP-mediated apoptosis, we hypothesized that AFP might interact with XIAP by displacing it from the complex with caspases, and thus preventing caspase inhibition We demonstrate here that AFP physically associates with XIAP in cytochrome c-activated cellular lysates, and that this complex does not contain the effector caspase We found that purified human AFP binds to recombinant XIAP, disrupts the association between XIAP and activated caspase 3, and antagonizes the antiapoptotic function of XIAP Our data indicated that AFP could also bind free XIAP to eliminate it from the reaction area and prevent caspase binding Results AFP promotes caspase activation in cell-free cytosolic extracts by blocking of XIAP-dependent inhibition Recent evidence has shown that AFP promotes the processing and activation of procaspase in the presence of low suboptimal doses of cytochrome c in cellfree cytosolic extracts Simultaneously, AFP induced the release of cIAP2 from the apoptosome complex [28] Our recent experimental data allowed us to hypothesize that AFP could operate as a XIAP antagonist by affecting the interaction of XIAP with active caspases, thus promoting their activity To determine whether AFP can affect caspase activity in HepG2 cytosolic extracts in the presence of an inhibitory amount of exogenous XIAP, we monitored caspase activation in a cell-free system Cytosolic cell extracts were activated by the addition of cytochrome c ⁄ dATP together with AFP or human serum albumin (HSA) in the presence of an inhibitory amount of rhXIAP Figure shows that addition of XIAP induced 50% inhibition of caspase activity in activated cell-free extracts Addition of AFP in the cytosolic extract induced significant enhancement of the DEVD-ase activity The same caspase activity was detected upon the simultaneous addition of an inhibitory amount of exogenous rhXIAP together with AFP The data clearly show that AFP abrogated the inhibitory activity of exogenous rhXIAP against endogenous caspases and failed to relieve AFP-mediated caspase stimulation By contrast, exogenous HSA did not affect caspase activity in cell-free extracts (Fig 1) Hence, AFP counteracts with XIAP by abrogation its caspase inhibition in the cytochrome c-activated cell-free cytosolic extracts AFP antagonizes XIAP function Fig AFP antagonizes XIAP-mediated caspase inhibition in cytochrome c-activated cell-free cytosolic extracts HepG2-derived cytosolic extracts were activated by mM dATP and lM cytochrome c and incubated with or without rhXIAP (250 nM) with the addition of 400 nM AFP or 400 nM HSA for h at 30 °C Control lysates incubated without addition of HSA and AFP were taken as controls Caspase activity was measured by DEVD–AMC cleavage The mean data in RFU ± SD from three independent experiments are shown AFP promotes caspase activity exclusively by abrogation of XIAP-dependent inhibition To study direct AFP ⁄ XIAP ⁄ caspase interaction we used recombinant proteins to form a reaction mixture in order to avoid the influence of other active compounds, which are available in cytosolic extracts An effective amount of rhXIAP was added into the solution of active recombinant caspase to induce 50% decrease of its activity The kinetics of the DEVD-ase cleavage in the reaction mixture was monitored each intervals rhXIAP in combination with HSA (Fig 2A) or alone (Fig 2B), induced twofold inhibition of caspase activity, but AFP ⁄ rhXIAP pretreatment significantly reduced inhibition by rhXIAP (Fig 2A) Addition of AFP or HSA alone did not affect caspase activity (Fig 2B) The results show that AFP does not directly affect caspase activity, but targets XIAP by blocking its inhibitory activity against caspase Therefore, AFP antagonizes XIAP function AFP competes with caspase and caspase for XIAP binding Functional interference of AFP and XIAP to examine their effect on caspase activity implied a direct physical interaction We further studied whether AFP can compete with caspases and for XIAP binding Pure recombinant His-tagged active caspases and were FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS 3839 AFP antagonizes XIAP function E Dudich et al Fig AFP abrogates XIAP-mediated inhibition of caspase activity in vitro Active recombinant caspase (3 nM) was treated with: (A) a mixture of rhXIAP (200 nM) with AFP (400 nM) or HSA (400 nM); (B) AFP (400 nM), HSA (400 nM), XIAP (200 nM) or without additions Caspase activity was measured by monitoring of DEVD–AMC cleavage at 5-min intervals Data were collected at 30 °C for 30 and expressed in RFU The mean ± SD of three independent determinations is shown incubated with rhXIAP and AFP or HSA, and protein complexes were thereafter immobilized on Ni–Sepharose beads After extensive washing the supernatant and pellets (beads) were blotted and probed with antibodies to XIAP AFP, but not HSA, completely abrogated the association of rhXIAP with caspases and (Fig 3, pellet) Western blotting revealed the presence of XIAP in the supernatants from Ni resin treated with AFP, but only a negligible amount of free rhXIAP was detected in the supernatants of HSA-treated samples (Fig 3) Western blotting of pellets using antibodies against caspase and caspase demonstrated that neither AFP nor HSA was able to modulate binding of His-tagged caspases on the nickel resin (Fig 3) The data clearly demonstrated that AFP cointeracted with XIAP by preventing XIAP ⁄ caspase complex formation AFP coprecipitates with endogenous XIAP in cellular extracts We further studied the ability of AFP to interact with endogenous XIAP in whole-cell extracts preactivated with cytochrome c ⁄ dATP ⁄ AFP Therefore, we examined whether AFP might be directly associated with XIAP or ⁄ and caspase in cell-free extracts Protein complexes were precipitated by the addition of corresponding antibodies and protein A–Sepharose beads Complex formation was detected by immunoblotting of the proteins bound to the protein A–Sepharose with anti-XIAP, anti-AFP, or anti-(caspase 3) IgG Figure shows that AFP coprecipitated with endogenous XIAP (Fig 4A, lane 3) but not with caspase (Fig 4C, lane 3840 Fig AFP prevents XIAP ⁄ caspases complex formation Human recombinant XIAP was incubated for h at °C with mixed His-tagged active recombinant caspase and caspase in the presence of AFP or HSA as described in Experimental procedures Protein complexes were precipitated by Ni–Sepharose beads Ni–Sepharose-bound proteins (pellet) and supernatants were analyzed by SDS ⁄ PAGE ⁄ immunoblotting with anti-XIAP, anti-(caspase 3) and anti-(caspase 9) sera Input 1: rhXIAP (100 ng); Input 2: recombinant His-tagged caspase (50 ng); Input 3: recombinant Histagged caspase (50 ng) 2; C, lane 3), whereas XIAP coprecipitated with both AFP and caspase (Fig 4A, lanes 2, 3) These results show that AFP associates physically with endogenous XIAP in activated cell cytosolic extracts (Fig 4A, FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS E Dudich et al AFP antagonizes XIAP function which was available in the cytosolic extracts (Fig 4A, lane 1), was not recovered on the immunoprecipitation ⁄ western blotting pattern with anti-(caspase 3) and anti-AFP IgG within the limits of detection (Fig 4A, lanes 2, 3) AFP physically associates with rhXIAP to form high molecular mass complexes A B C Fig AFP associates physically with endogenous XIAP in the cellular cytosolic extracts HepG2 cytosolic extract was activated by the addition of lM cytochrome c and mM dATP for 30 at 30 °C in the presence of AFP (6 lg) and thereafter the specific interaction of AFP, XIAP and caspase was tested using coimmunoprecipitation with anti-AFP, anti-(caspase 3) or anti-(rabbit IgG) as negative control Western blot analysis was carried out with antiXIAP (A), anti-AFP (B) and anti-(caspase 3) (C) sera Input: cytosolic extract activated with cytochrome c ⁄ dATP (20 lg) Molecular mass markers are indicated on the left lane 3) As expected, endogenous caspase coprecipitated with endogenous XIAP (Fig 4A, lane 2) No interaction between caspase and AFP could be detected (Fig 4B, lane 2; Fig 4C, lane 3) This indirectly proved that AFP and caspase interacted with the same binding site of XIAP If AFP had been attached to a binding site on the XIAP molecule other than that responsible for caspase binding, we would be able to detect coprecipitation of all three proteins in this experiment Hence, the results showed that AFP actively binds to endogenous XIAP in cytochrome c-activated cellular extracts, but that it also prevents complex formation of XIAP with active caspases The data also suggested that AFP seems to bind to the entire XIAP molecule only, because fragmented XIAP, We then determined whether AFP and XIAP were able to form intermolecular complex rhXIAP was coincubated with rhAFP and then the protein mixture was subjected to native electrophoresis The complex formation was analyzed by western blotting with antiAFP IgG (Fig 5A, lanes 1, 2) and with anti-XIAP IgG (Fig 5B, lanes 1, 2) Probing with anti-AFP revealed three AFP-specific bands (Fig 5A, lane 2), which correspond to the AFP-monomer, natural AFPdimer and high molecular mass upper band corresponding to the AFP-specific macromolecular complex To identify presence of XIAP in the AFP-specific complexes, we probed the blot pattern with anti-XIAP IgG This revealed presence of XIAP in the upper band, corresponding to the high molecular mass AFPspecific complex (Fig 5B, lane 2) We conclude that incubation of pure AFP with XIAP led to the formation an intermolecular complex This AFP ⁄ XIAP complex evidently contains more than two proteins, showing the ability of AFP to form multimolecular high-affinity complexes with XIAP A B Fig Direct AFP ⁄ XIAP complex formation in vitro Recombinant human AFP and XIAP were coincubated for h at °C and thereafter subjected to the native nondenaturing PAGE followed by western blotting with anti-AFP (A) and anti-XIAP (B) IgG Lane on the each pattern corresponds to rhAFP alone and lane corresponds to AFP ⁄ XIAP FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS 3841 AFP antagonizes XIAP function E Dudich et al Search for the potential IBM in the structure of the AFP molecule The AFP protein contains a putative IBM-like sequence ATIF(29–32), which fits the IAP binding tetrapeptide consensus [9,44] (Fig 6) Similar to the other IBM proteins, Smac, Omi and GSPT1 [17–19], AFP requires N-terminal processing to expose the IBM motif at the newly generated N-terminus Processing of the first 28 amino acids could allow exposing of N-terminal motif ATIF that is highly reminiscent of IBM of caspase and other IAP antagonists (Fig 6) In common with other IBMs, the IBMlike motif of AFP bears Ala at its N-termini The Ala residue within the IBM is highly conserved (Fig 6) and has been shown to be essential for the interaction between XIAP and mature Smac ⁄ Diablo [45] This sequence displays a high degree of similarity to the IBM of caspase with a single replacement of Pro3 in caspase to Ile3 in AFP (Fig 6) [22] This position is variable in different IAP antagonists and does not seem to be critical in forming the XIAP-binding site (Fig 6) The AFP IBM-like sequence has Phe at the P5 position, as in Drosophila Sickle and Grim and Xenopsis Casp-9 (Fig 6) [22,46] As shown previously [46], Phe at P5 position is clearly favored for BIR2 binding The requirement for proteolytical processing of AFP to expose the N-terminal IBM may explain why only part of the total amount of AFP, that which has undergone proteolytical processing, participates in complex formation with XIAP (Fig 5A) Proteolytical processing of AFP is usually observed in cytochrome c-activated lysates [28] It has been shown that proteolytical cleavage of pure AFP results in AFP fragments exposing different destabilizing N-terminal residues [27] However, our results show that pure recombinant AFP and XIAP can interact without any requirements for the presence of active caspases in the reaction mixture We tentatively suggest the existence of another IAP-binding site in AFP, one which does not require N-terminal processing to be activated for XIAP binding Structural modeling of the AFP(dimer) ⁄ BIR2–3 complex Our results show that AFP can bind to an entire XIAP molecule but not to its fragments It has also been demonstrated that AFP displaces caspase from its complex with XIAP, suggesting that the BIR2 domain is involved in this interaction The data also suggest that AFP uses at least two different XIAP binding sites to form the AFP–XIAP complex Previous studies suggest that AFP [27], as well as XIAP [47], is able to Fig Sequence alignment of IBM-bearing proteins Collinear alignment of the N-terminal sequence 1–55 from HSA and 1–60 from human AFP (upper) Sequence alignment of IBM-bearing proteins: human AFP, caspase 9–p12, caspase 7–p20, Smac ⁄ DIABLO, GSPT1; Omi ⁄ Htr2; mouse caspase 9–p12; Xenopus caspase 9–p12; Drosophila ICE, Reaper, Grim, Hid, Sickle, Jafrac2, GSPT1; C elegans GSPT1 Identical residues are highlighted in black Residues conserved in several IBM proteins are indicated in grey IBM-like sequence is boxed Protein sequence data have been taken from the Protein Data Bank [63] 3842 FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS E Dudich et al dimerize, which could create many possibilities for interaction stoichiometry We suggest that AFP dimer forms a complex with XIAP by interacting with both BIR2 and BIR3 domains The involvement of BIR3 in the interaction with AFP is supported by recent studies showing that AFP induced the release of both caspase and caspase 9, as well as cIAP-2, from the apoptosome complex [28] It is possible that caspase cointeracts with AFP ⁄ XIAP complex similarly to caspase It is expected that AFP dimer interacts with the BIR2 and BIR3 domains of XIAP and forms a : stoichiometric complex Native electrophoresis indicates that the AFP ⁄ XIAP complex is formed by more than two molecules and includes at least three members (Fig 5) Taking into account that both AFP and XIAP tend to dimerize, a few interaction models can be proposed We suggest a simple complex composed of AFP dimer and XIAP monomer The 3D molecular structure of the AFP molecule remains unsolved Human AFP and HSA exhibit 39% amino acid sequence homology [44] The authentic structural homology of AFP and HSA allowed us to predict the tertiary structure of AFP based on the atomic coordinates obtained by X-ray crystallography for HSA [48] Using the NMR structures of the BIR2 [49] and BIR3 [50] domains, the AFP(dimer) ⁄ BIR2–3 complex was constructed (Fig 7) In this complex, the Fig Hypothetical molecular model of the AFP(dimer) ⁄ BIR2–3 complex Each monomer of the AFP dimer is shown in blue and red, respectively The BIR2 and BIR3 domains are shown in green The dashed yellow lines connect the ATIF peptides of each AFP monomer and the IBM-interacting grooves of the BIR2 and BIR3 domains The figure was produced using MOLSCRIPT v 2.1 [62] AFP antagonizes XIAP function BIR2 and BIR3 domains show the same local twofold symmetry as two AFP monomers in the AFP dimeric structure Moreover, the IBM-interacting grooves of the BIR2 and BIR3 domains lie close to the ATIF-end of the first and second AFP monomers, respectively, allowing for the possibility that they belong to the same XIAP molecule Discussion Recent evidence has broken the main paradigm of apoptosis, stating that the release of cytochrome c is the point of no return in the apoptotic program [17,18,51,52] It has been shown that certain tumor cells are able to recover after cytochrome c release and survive despite the constitutive presence of cytochrome c in the cytosol in the absence of any signs of apoptosis [53] Moreover, caspase activation does not always result in cell death [16–18,52] The ability of the cell to die at the postmitochondrial level depends mainly on the activity of endogenous inhibitors of apoptosis, such as IAPs, sHSPs, or Bcl-2 [6,7,54,55] There is further evidence of a high level of apoptotic activation and the upregulation of IAPs in tumor tissue [8,16] Inactivation of XIAP or the cancellation of XIAP inhibition appears both necessary and sufficient for cytochrome c to activate caspases and trigger cell death [9,16] The activity of IAPs is regulated by a group of IAP-regulatory proteins that bind to IAPs and inhibit their antiapoptotic function [17–21] These factors are important research targets to search for new nontoxic drugs with selective pro-apoptotic activity for tumor cells The identification of protein drugs, which can overcome the tumor defense system by preventing the realization of apoptosis in tumor cells, will have a great potential as tumor therapeutic agents [9] In this study we showed that AFP could participate in the regulation of apoptosis in tumor cells by counteraction with the most potent endogenous inhibitor of mammalian caspases XIAP Our results show that AFP binds to XIAP and disrupts its interaction with caspase These results are in harmony with the fact that AFP can bind to cIAP-2 and disrupt its interaction with caspase and caspase [28] Because pure AFP can bind XIAP in vitro, this interaction appears to be direct The binding seems to be highly specific, because it did not occur with the nonapoptotic protein HSA, structure and function of which is closely related to AFP In addition to the direct association with XIAP, AFP could also relive the XIAP-inhibitory effect on the activity of the mature recombinant caspase Moreover, AFP shows the ability to enhance FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS 3843 AFP antagonizes XIAP function E Dudich et al cytochrome c-dependent activation of caspase and caspase in the presence of an inhibitory amount of exogenous XIAP In this respect, AFP behaves in a similar manner to the IAP antagonists This group of proteins is characterized by the presence of the N-terminal conserved BIR-binding motif (IBM), which is required for IAP binding [20] The presence of IBM in the intracellular protein allowed us to recognize its possibility of serving as an IAP antagonist [9,13,23] Mammalian proteins Smac ⁄ DIABLO, Omi ⁄ HtrA2 and GSPT1 ⁄ eRF3 are released from mitochondria upon triggering apoptosis and require processing to reveal the IBM at the newly generated N-terminus [17–19] However, other proteins, such as ARTS and XAF1, which not contain an IBM-like motif, were also seen to antagonize IAP function via an unknown mechanism [20,21] The search for the potential IBMlike sequence in the structure of AFP revealed the presence of a similar amino acid sequence ATIF in human AFP at position 29–32 [44] It can be proposed that processing of the first 28 amino acid residues generates the AFP fragment with an N-terminal motif ATIF that is highly reminiscent of IBM of caspase and other IAP antagonists (Fig 6) Our results indicate that only entire XIAP could bind AFP (Fig 5) This means that AFP binds simultaneously to at least two BIR domains BIR3 binding is preferential for an IBM-like motif similar to that available in caspase Taking into account that AFP competes with caspase for complex formation with XIAP, we consider that both BIR2 and BIR3 may be involved in complex formation with AFP A similar model has been described previously [56] for a complex of dimeric Smac protein with recombinant XIAP fragments containing both BIR2 and BIR3 domains, or for a complex of XIAP with active caspases and -7 [10] Another model, which involves the entire AFP molecule, could be also proposed, and will introduce other parts of the molecule in their interaction with XIAP To identify the molecular mechanism of the AFP ⁄ XIAP interaction additional structural studies are needed Although our results show that AFP interacts physically with XIAP and protects activated caspases from IAP-induced inhibition, they not reveal how it operates There are several possibilities However, a functional preference of AFP for tumor cells seems evident [30–41] It has previously been shown that AFP selectively penetrates tumor cells via specific membrane AFP receptors expressed on the surface of tumor cells but not on normal adult cells [32–36] Unlike other anti-IAPs, such as Smac or Omi, which have an exclusively mitochondrial localization and become available 3844 to interact with IAPs only after apoptosis has been triggered by cytochrome c release, AFP was available whenever it entered into the cell via cellular membrane receptors or was synthesized inside the cell Thus, AFP is able to regulate the IAP level in the cytosol independently of whether cell is undergoing apoptosis or not Under conditions of constitutively high levels of XIAP expression in tumor cells [57], AFP could reduce its protein level, presumably by proteosome-mediated degradation Comparison with normal cell lines and tissues has shown that many tumor cell lines and tissues have constitutively higher levels of active caspase and free cytochrome c in the absence of apoptotic stimuli and yet are not undergoing apoptosis [58,59] Simultaneously, tumor cells have high levels of expression of survivin and XIAP [57,60] Survival of cancer cells is possible under conditions of pacific equilibrium between pro- and antiapoptotic signals Taking into account that normal cells and tissues not overexpress apoptotic stimuli and IAPs, whereas cancer cells and tissues do, IAP-targeting drugs will have highly selective proapoptotic activity for cancer cells and little toxicity towards normal cells [8,9,16] The general obstacle preventing the design of apoptosis-regulating drugs on the basis of known natural anti-XIAPs is their intracellular localization and the inability to use them as internal regulating factors Considering that AFP can penetrate selectively into tumor cells via specific membrane receptors, the molecular mechanism of AFP-mediated targeting regulation of apoptosis could be suggested to be as follows: (a) AFP selectively penetrates tumor cells via specific AFP receptors; and (b) formation of the AFP–XIAP complex prevents its binding to activated caspases, increases XIAP instability against ubiquition ⁄ proteasomal destruction and reduces the XIAP level to promote apoptosis induction This function of AFP may serve to sensitize tumor cells to weak proapoptotic stimuli by inducing a tumor-specific response to chemotherapeutic or radiotherapeutic treatments The selectivity of the AFP-mediated proapoptotic activity for tumor cells may be explained by its counteraction with IAPs, which are shown to be dominantly overexpressed in tumor cells under conditions of the simultaneous existence of high levels of various active proapoptotic factors Normal cells not undergo AFP-induced apoptosis because they not express high levels of IAPs, not contain constitutively activated caspases and not express membrane AFP receptors AFP seems to be directly involved in targeting positive regulation of the apoptotic pathway dysfunction in cancer cells by FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS E Dudich et al inhibition of IAP function leading to triggering of the apoptosis machinery Further studies are required to better understand the importance of the role AFP in modulating the level of IAPs in tumor cells Elucidation of the role of AFP in tumor cell-specific regulation of XIAP function in apoptosis may have important implications for cancer treatment and prevention AFP and AFP-derived peptides can potentially be used to overcome drug resistance caused by the differential mechanism of apoptosis dysfunction in cancer cells Experimental procedures Purification of a-fetoprotein Embryonic AFP was isolated from human cord serum using ion-exchange, affinity and gel-filtration chromatography as described previously [27] The purity and homogeneity of the protein were assessed by SDS ⁄ PAGE and western blotting with AFP-specific polyclonal antibodies as described elsewhere [28] Recombinant human AFP (rhAFP) was purified from the culture medium of recombinant Saccharomyces cerevisiae as described previously [43] using affinity and gel chromatography Cells HepG2 cells originating from the American Type Culture Collection were grown in Dulbecco’s modified Eagle’s medium (ICN Biomedicals, Inc., Costa Mesa, CA) with addition of l-glutamine, 10% heat-inactivated fetal bovine serum, penicillin (100 unitsỈmL)1), streptomycin (0.1 mgỈmL)1) in a humidified 5% CO2 atmosphere at 37 °C For a passage, cells were incubated in 0.25% trypsin solution, then washed and plated out Preparation of cell-free cytosolic extracts Cell-free cytosolic extracts were generated from human hepatocarcinoma HepG2 as described previously [60] with minor modifications [28] Cells (4 · 108) were collected and washed three times with 50 mL NaCl ⁄ Pi and once with mL hypotonic cell extraction buffer (CEB; containing 20 mm Hepes, pH 7.2, 10 mm KCl, mm MgCl2, mm dithiothreitol, mm EGTA, 25 lgỈmL)1 leupeptin, lgỈmL)1 pepstatin, 40 mm b-glycerophosphate, mm phenylmethylsulfonyl fluoride) The cell pellet was then resuspended in an equal volume of CEB, allowed to swell for 20 on ice and then disrupted by passing through a needle The homogenate was centrifuged at 5000 g for 10 at °C to remove whole cells and nuclei Thereafter the supernatant was centrifuged at 15 000 g for 20 at °C The procedure was repeated twice Cytosolic extracts were AFP antagonizes XIAP function assessed for protein content by Bradford assay and stored in aliquots at )70 °C Analysis of caspase activity Caspase assays were performed with active recombinant caspase-3 (Alexis Corp., San Diego, CA), recombinant fulllength rhXIAP (R&D Systems, Minneapolis, MN), purified AFP, and HSA (Sigma-Aldrich, St Louis, MO) All other reagents were from Sigma, unless stated otherwise RhXIAP (200 nm) was incubated with AFP (400 nm) or HSA (400 nm) in IAP buffer (50 mm Hepes, pH 7.5, 100 mm NaCl, mm EDTA, mm dithiothreitol, 0.1% Chaps, 10% sucrose) for 15 at room temperature Thereafter the active recombinant caspase (3 nm) was added to the reaction mixture, and incubation continued for a further 15 under the same conditions For the control, caspase was incubated with each of the following compounds separately: AFP (400 nm), HSA (400 nm) or XIAP (200 nm) The kinetics of caspase activity was monitored by cleavage of the fluorogeneic substrate [50 lm Ac-Asp-Glu-Val-Asp-7-amino-4-methyl coumarin (Ac-DEVDAMC), Sigma] at 5-min intervals for 30 To assess the effects of AFP, HSA and XIAP on caspase activity in cellular extracts in vitro, rhXIAP (250 nm) was incubated with cytosolic extract (40 lg) activated by addition of lm of bovine heart cytochrome c and mm dATP in the presence of AFP (400 nm) or HSA (400 nm) in 15 lL of a reaction buffer (10 mm Hepes, pH 7.2, 25 mm NaCl, mm MgCl2, mm dithiothreitol, mm EDTA, 0.1 mm phenylmethylsulfonyl fluoride) for h at 30 °C, and the reaction mixtures were analyzed for Ac-DEVD-AMC cleavage Caspase activity was determined by adding lL of cell extracts to 16 lL of substrate reaction buffer (20 mm Hepes, pH 7.2, 100 mm NaCl, 10 mm dithiothreitol, mm EDTA, 0.1% Chaps 10% sucrose, 50 lm Ac-DEVD-AMC) for 40 at 30 °C The reaction was stopped by the addition 200 lL of cold NaCl ⁄ Pi, and AMC liberation was measured using Victor-1420 Multilabel counter (Wallac, Finland) at excitation 355 nm and emission 460 nm All samples were analyzed in duplicate and the experiments were repeated three times For each sample, caspase activity was expressed in relative fluorescent units (RFU), showing the amount of cleaved substrate normalized for protein content Direct protein–protein binding assay To determine possible interactions between AFP, caspase 3, caspase 9, and XIAP, we used a direct coprecipitation assay with purified proteins Human recombinant XIAP (350 ng), His-tagged human recombinant caspase (50 ng), anf active His-tagged rat recombinant caspase (300 ng) were mixed with 0.5 mgỈmL)1 AFP or 0.5 mgỈmL)1 HSA in FEBS Journal 273 (2006) 3837–3849 ª 2006 The Authors Journal compilation ª 2006 FEBS 3845 AFP antagonizes XIAP function E Dudich et al (Amersham Pharmacia Biotech) according to manufacturer instructions 15 lL buffer A (20 mm Hepes, pH 7.4, 100 mm NaCl, 0.5 mm EDTA, 0.5 mm dithiothreitol, 0,1% Chaps, 10% sucrose) and incubated for h at °C Thereafter, 15 lL of Ni-Sepharose beads (Qiagen, Valencia, CA) in 80 lL of buffer B (20 mm Na2HPO4, pH 7.2, 0.2 m NaCl) were added to the reaction mixture and incubation was continued for the next h The beads were separated from supernatants by centrifugation and both fractions were collected Protein–bead complexes were washed four times and boiled in 20 lL of reducing · Laemmli sample buffer Samples, protein–beads and supernatants were analyzed by SDS ⁄ PAGE ⁄ immunoblotting in 12.5% polyacrylamide gel in with b-mercaptoetanol To study direct AFP–XIAP protein interaction, human recombinant XIAP (1.5 lg) and human recombinant AFP (2 lg) were incubated for h at °C in 30 lL of buffer C (20 mm Hepes, pH 7.2, 140 mm KCl, mm MgCl2, mm dithiothreitol, mm EDTA, 0.1% OVA) Thereafter, protein mixtures (5 lL) were subjected to 8% nondenaturing continuous polyacrylamide gel (Tris pH 8.7) and separated by Native electrophoresis [61] In order to predict the tertiary structure of the AFP molecule [44] the molecular modeling software package sybyl was used (Tripos Associates, Inc., St Louis, MO) A model of the dimeric structure of the AFP was reconstructed by using of the atomic coordinates obtained by X-ray crystallography for HSA [48] and plotted using molscript v 2.1 [62] The atomic coordinates of HSA (code 1AO6) were obtained from the Protein Data Bank [63] The primary structure alignment of AFP and HSA was constructed using multalin [64] The model of AFP was minimized to ˚ an energy gradient < 0.050 kcalỈmol)1ỈA)1 using the Tripos force field and a combination of Simplex [65] and Powell algorithms [66] Coordinates of the NMR structures of the BIR2 [49] and BIR3 [50] domains were achieved from the Protein Data Bank files: 1C9Q and 1F9X, respectively Immunoprecipitation Acknowledgements Cytosolic extracts obtained from HepG2 cells were normalized for protein content (500 lg of total protein in 100 lL of buffer C) and activated by addition of lm cytochrome c and mm dATP for 30 at 30 °C in the presence of AFP (6 lg) The reaction mixtures were cooled and incubated with lg of the following antibodies: polyclonal rabbit anti-AFP [28], normal rabbit IgG (Sigma), or with rabbit anti-(caspase 3) (Santa Cruz, Santa Cruz, CA) for h with a gentle mixing at °C Thereafter, 40 lL of protein A–Sepharose bead slurry (Amersham Pharmacia Biotech) were added to the each sample Samples were incubated overnight in a rotating shaker at °C The beads were pelleted by centrifugation and after intensive washing, were syringe dried The bound proteins were eluted by boiling in 25 lL of 2· sample buffer Samples in aliquots of 10 lL were loaded onto the 12.5% SDS polyacrylamide gel and subjected to SDS ⁄ PAGE ⁄ immunoblotting This work was supported in part by the International Science & Technology Center, ISTC (grant #1878); by the Academy of Finland (Grant # 107762), the Neobiology program of the Technology Development Center ´ for Finland (TEKES), and the Sigrid Juselius Foundation Immunoblotting analysis Samples after SDS ⁄ PAGE or native PAGE were electroblotted onto a poly(vinylidene 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