2Inhibitor of Apoptosis Proteins

Inhibitor of apoptosis proteins (IAPs) constitute a family of apoptosis-suppressing proteins that contain at least one copy of a conserved domain called baculoviral IAP repeat (BIR), a zinc-binding fold involved in protein interactions. Humans and other mammals contain multiple genes encoding IAP family members, providing a diversity of variants with both common and specialized functions. IAPs are known for their ability to bind certain caspases, which are proteases responsible for apoptosis. Several IAPs contain RING (really interesting new gene) domains that bind ubiquitin-conjugating enzymes (UBC), whereas others possess UBC catalytic domains. These attributes endow many IAPs with E3 ligase activity, implicating them in the ubiquitinylation and proteasome-dependent degradation of a variety of cellular substrates. In addition, several IAP family members have multifaceted functions as platforms for coordinating signal transduction events associated with activation of particular protein kinases. Finally, some IAPs have dual functions as regulators of cell death and cell division. In this chapter, we provide an overview of IAP family proteins, including their structures and domain organizations, biochemical and cellular functions, intracellular locations, post-translational modifications, and relevance to disease.

1. THE BIR DOMAIN DEFINES MEMBERSHIP

IN THE IAP FAMILY

The IAPs are structurally defined by their BIR domains and functionally defined by their ability to block apoptosis. The evolutionarily conserved BIR domains are located at the N-terminus of all IAP family members and are present as a single copy or in groups of two to three tandem repeats. BIR domains are composed of 70 amino acids and contain the signature

sequence CX2CX16HX6C. Each BIR domain folds as a three-stranded β-sheet with four to five α-helices, which pack tightly to form a hydrophobic core. The BIR structure is stabilized by a single zinc molecule coordinated by three cysteines and a histidine (Figure 2-1). BIR domains mediate protein–protein interactions among themselves and other proteins. However, not all BIRcontaining proteins are apoptosis suppressors in all species in which they occur. The roles of some mammalian BIR-containing protein in apoptosis inhibition, for example, may be indirect or of questionable physiologic relevance, and BIR-containing proteins of some lower organisms (e.g., yeast, worms) most certainly are unrelated to control of apoptosis.

In humans, eight genes encoding BIR-containing proteins have been identified (Figure 2-2), and all of these have been reported to suppress apoptosis – at least when over-expressed in cultured cells. The human IAPs include Apollon (BRUCE; BIRC6), cellular IAP1 and IAP2 (c-IAP1/c-IAP2; BIRC2 and BIRC3), IAP-like protein 2 (ILP-2)/testis-specific IAP (Ts-IAP; BIRC8), Livin/melanoma IAP (ML-IAP; BIRC7), neuronal apoptosis inhibitory protein (NAIP; BIR1), Survivin (BIRC5), and X-linked IAP (XIAP; BIRC4). Orthologs of all eight of the human IAPs are found in mice; however, mice have an expanded NAIP locus that is highly polymorphic among mouse strains and that contains up to three copies of the gene, wherein several functional copies of NAIP are often expressed under different promoters.

Survivin (BIRC5) is the smallest of the human IAPs, containing a single BIR followed by a coiled-coil domain that contributes to dimerization of this protein. Livin (BIRC7) and ILP-2 (BIRC8) are slightly larger, containing a single BIR followed by a RING domain. The RING domain is characterized by the presence of six to seven cysteine residues and one to two histidines that

Figure 2-1. 3D Structure of XIAP BIR3. Ribbon depiction of the structural of BIR3 domain of XIAP (residues 255–346). The α-helices are shown in red, β-sheets are shown in green, zinc is shown in purple, and the side chains of the residues that chelate zinc are shown in yellow. Structure adapted from Sun et al. (2000) J Biol Chem 275:33777– 81. C 2000 The American Society for Biochemistry and Molecular Biology. See Color Plate 1.

coordinate two zinc ions. This domain imparts E3 ubiquitin ligase activity on many proteins by virtue of its ability to bind UBCs. Apollon (BIRC6) also contains a single BIR domain but is a huge protein, containing a large C-terminal domain that contains a UBC catalytic domain. NAIP, c-IAP1, c-IAP2, and XIAP contain three

tandem copies of the BIR domain. In c-IAP1, c-IAP2, and XIAP, the BIR domains are followed by a ubiquitinassociated (UBA) domain and a RING domain. These proteins also have E3 ligase activity. In addition, c-IAP1 and c-IAP2 contain a caspase activation and recruitment domain (CARD) (Figure 2-2), presently of unknown function. Interestingly, however, many proteins involved in either apoptosis or innate immunity contain CARDs. The three BIR domains of NAIP are followed by a NACHT domain (homologous to the nucleotide-binding oligomerization domains of Nod-like receptor [NLR] family proteins), followed by several leucine-rich repeat (LRR) domains. In NLR family proteins, the LRRs bind pathogen-derived molecules, an event required for rendering NACHT domains capable of binding nucleotides and oligomerizing.

Marine organisms vary greatly in their BIR-encoding genes. The vertebrate fish species Danio rerio (zebrafish) contains four genes encoding BIRs, where the BIR is found associated with CARD, RING, and UBC domains, similar to land vertebrates. Similarly, the marine invertebrates Ciona intestinalis (ascidian) and Strongylocentrotus purpuratus (sea urchin) have at least three and two BIR-encoding genes, respectively. In these organisms, the BIR is found in association with CARD, RING, and UBC domains, similar to land animals.

The genome of the fruit fly, Drosophila melanogaster, contains four BIR-encoding genes with varying

Table 2-1. Summary of human IAP family members and their various functions

functions: DIAP1 (Drosophila IAP-1), DIAP2, deterin, and dBruce. DIAP1 (Drosophile IAP1) contains two BIR domains and a C-terminal RING domain. DIAP2 contains three BIRs and a RING domain. Deterin is a small, Survivin-like IAP. Finally, dBruce (the Drosophila ortholog of Apollon/BRUCE), contains a single BIR domain as well as the UBC domain, similar to its counterparts in mammals. The nematode Caenorhabditis elegans contains two gene-encoding BIR proteins: BIR-1 and BIR-2. The yeasts Schizosaccharomyces pombe and

Saccharomyces cerevisiae each produce a Bir1p protein with two tandem BIR domains. Bir1p acts similarly to mammalian Survivin in that it helps regulate the cell cycle (Figure 2-3).

2. CELLULAR FUNCTIONS AND PHENOTYPES OF IAPS

The cellular functions of IAPs include regulation of apoptosis but extend beyond cell death control – presumably reflecting the dual role that some of these proteins play in a variety of cellular processes. Table 2-1 provides a summary of the varying functions of the eight human IAP family members. All human IAPs are capable of

reducing apoptosis when over-expressed by gene transfection in cultured cells. Gene silencing by antisense oligodeoxynucleotides (AS-ODNs) or small interfering RNAs (siRNAs) has demonstrated a requirement for the IAP family members XIAP, c-IAP1, c-IAP2, ML-IAP, Livin, Apollon, and Survivin either for survival in culture or for resistance to certain apoptotic stimuli among various tumor cell lines. Over-expression of IAPs blocks apoptosis induced via the extrinsic pathway (tumor necrosis factor [TNF] family death receptors), intrinsic pathway (mitochondria-initiated), or both, depending on the specific IAP and the cellular context. For example, XIAP suppresses both extrinsic and intrinsic pathways, whereas Survivin, Livin, and ML-IAP have been reported to preferentially or exclusively inhibit the intrinsic pathway.

Certain BIR domain-containing proteins regulate cell division. In this regard, Survivin plays a role in chromosome segregation and cytokinesis, displaying a pattern of expression different from other IAPs in that it is expressed at high levels in embryonic tissues and in transformed cells. Survivin is not expressed in normal interphase cells of mammals, but its expression increases markedly during G2-M phase of the cell cycle in dividing cells. Survivin is required for proper chromosome segregation at the metaphase to anaphase

Figure 2-3. Comparison of BIR domains. Phylogenetic relationship of the IAP family of proteins is presented based on the sequences displayed in the MegAlign (DNASTAR) document using the CLUSTAL method. Full-length human BIR domain containing proteins: Livin, c-IAP1, c-IAP2, Ts-IAP, XIAP, Apollon, survivin, and NAIP. Drosophila melanogaster proteins: DIAP1, DIAP2, deterin, and BRUCE. Caenorhabditis elegans proteins: BIR-1 and BIR2. Yeast proteins: Schizosaccharomyces pombe (sp)Bir1p and Saccharomyces cerevisiae (sc)Bir1p.

transition and is essential for cytokinesis during telophase, when replicated daughter cells split. Similar roles have been reported for the Survivin ortholog of flies (deterin) and the BIR family proteins of worms (C. elegans) and yeast (S. cerevisiae; S. pombe) with respect to both mitosis and meiosis, suggesting an ancient role for these BIR-containing proteins in cell division. For example, S. cerevisiae Bir1p null mutant strains display instability of the yeast mini-chromosome, a chromosome mis-segregation phenotype.

Several IAPs play roles in signal transduction, as described in Section 7 in more detail. The signaling pathways affected by IAPs include nuclear factor kappa B (NF-κB) and stress kinases (c-Jun N-terminal kinase [JNK]; p38 mitogen-activated protein kinase [MAPK]). XIAP has been identified as a critical component of signaling by certain bone morphogenic protein (BMP) receptors, such as BMP type I. The c-IAP1 and c-IAP2 proteins have been implicated in signal transduction by certain TNF family receptors.

NAIP is unique among mammalian IAPs in that it is both a BIR-containing IAP and also a member of the NLR family of innate immunity receptors (containing NACHT and LRR domain), which function in host–pathogen responses. The LRRs of NAIP are thought to operate as an intracellular sensor (receptor) of pathogens, in particular, bacterial flagellin. Evidence has shown that NAIP becomes activated on exposure to flagellin, resulting in activation of caspase-1 – a protease that cleaves and activates proinflammatory cytokines, pro- interleukin-1β (pro-IL-1β), pro-IL-18, and pro-IL-33. In Drosophila, the IAP family member DIAP2 is required for expression of endogenous antimicrobial peptides (AMPs), highlighting the role of this protein in innate immune responses. DIAP2 null flies infected with Gramnegative bacteria fail to mount an immune response and die. Recently, XIAP, cIAP1, and cIAP2 were also implicated in innate immunity by virtue of their role in signaling by NLR family members NLRC1 (NOD1) and NLRC2 (NOD2) [see section 7 for more detail]. Thus, connections between IAPs and innate immunity are robust – a feature often found in apoptosis-regulating proteins.

Proper copper homeostasis is essential to avoid toxic effects of excessive copper levels. Proteins that facilitate this balance work to export excess copper from cells, such as copper metabolism gene MURR1 domain containing 1 (COMMD1). XIAP is a rate-limiting component in determining intracellular copper concentration. XIAP directly binds copper and COMMD1 to mediate the ubiquitinylation and proteasomal degradation of COMMD1. Copper binding results in an apparent conformational change in XIAP, rendering it unstable and susceptible to proteasomal degradation.

3. IN VIVO FUNCTIONS OF IAP FAMILY PROTEINS

IAP family genes have been ablated in mice for six of the eight mammalian family members. Organism-wide gene ablation is embryonic lethal for Survivin (before E4.5) and BRUCE (E14.5 to the perinatal stage). Knockouts of NAIP, c-IAP1, c-IAP2, and XIAP, in contrast, show no overt phenotype, but detailed examination reveals specific attributes. Mice with complete ablation of NAIP exhibit normal development yet show increased susceptibility to seizure-induced cell death. XIAP knockout mice display delayed lobuloalveolar development in the mammary gland yet no altered apoptotic sensitivity. Overall, however, mouse gene knockout studies for IAP family members NAIP, c-IAP1, c-IAP2, and XIAP have failed to produce blatant cell death phenotypes, suggesting perhaps that redundancy among IAP family members ensures cell survival during normal development and adult tissue homeostasis.

In addition to knockout mice, a variety of IAPs have been over-expressed in transgenic mice. Transgenic mice over-expressing c-IAP2 in the heart show resistance to apoptosis and cardiac dysfunction after ischemia/reperfusion injury. Using a cochlea-specific promoter to over-express XIAP in the inner ear, hearing and hair cell loss in the cochlea were reduced when compared with control mice. Additionally, high levels of human XIAP mRNA expression are present in developing T cells of the thymus and peripheral lymph nodes, and transgenic mice over-expressing an XIAP transgene (under the control of a T-cell–specific promoter) exhibit accumulation of thymocytes and/or T cells in primary (thymus) and secondary (spleen) lymphoid tissues, providing evidence that XIAP plays a role in the homeostatic balance of lymphocyte populations.

Although complete ablation of Survivin is embryonic lethal, a number of conditional knockout models exist, emphasizing the important role of this protein in normal physiologic development. Early deletion of Survivin in thymocytes of mice shows pre–T-cell receptor proliferation checkpoint arrest, whereas its loss at later stages results in normal thymic development, with neither leading to an increase in apoptotic sensitivity. In neuronal precursor cells, conditional deletion of Survivin (at day E10.5) leads to massive apoptosis, and affected neonates die shortly after birth as a result of respiratory insufficiency.

In zebrafish, knockdown of Survivin using AS-ODN (based on morpholino chemistry) in embryos results in reduced eye and head size and also causes defective angiogenesis. In addition, zebrafish with null mutations in c-IAP1 display abnormal vascular development, further suggesting a role for IAPs in angiogenesis