EvoprintHD of thread

BIOLOGICAL OVERVIEW

The inhibitor of apoptosis (IAP) proteins are conserved from yeast to humans and are also found in certain viruses that infect invertebrates, including baculoviruses and entomopoxviruses. There are two family members in Drosophila: Thread, also know as DIAP1, and Inhibitor of apoptosis 2 (Iap2). IAP proteins are identified by the presence of 1-3 copies of a motif called a baculovirus IAP repeat (BIR) at the amino terminus. Many IAP proteins also contain a RING finger motif at the carboxyl terminus, some of which have been shown to possess E3 ubiquitin ligase activity. Thus some IAP proteins, including Thread, function in the ubiquitin pathway to enhance protein degradation. Most IAP proteins from insects and vertebrates are capable of inhibiting apoptosis when overexpressed, whereas IAP homologs in nematodes and yeast instead play a role in regulating cytokinesis (Salvesen, 2002; Muro, 2002 and references therein).

IAP proteins inhibit apoptosis stimulated by a variety of signals in both insect and mammalian cells, presumably at least in part through their ability to inhibit caspases, a family of cysteine proteases that mediate many of the morphological and biochemical changes associated with apoptosis. Following a death signal, apical or signaling caspases become activated through proteolytic processing. These apical caspases in turn proteolytically activate other caspases, called effector caspases, that go on to cleave various target proteins, leading to apoptosis. In mammalian cells, two major pathways leading to apical caspase activation have been described, the extrinsic and intrinsic pathways. The extrinsic pathway primarily involves the activation of the apical caspases caspase-8 and -10 by death receptors such as fas and tumor necrosis factor receptor, whereas the intrinsic pathway involves the release of factors from mitochondria such as cytochrome c that results in the activation of apical caspase-9 via the Apaf-1 protein (see Drosophila Apaf-1-related-killer), an oligomerizing factor required for the activation of caspase-9 in mammals. Upon binding to cytochrome c, Apaf-1 forms large oligomeric complexes known as apoptosomes that recruit and activate caspase-9. In mammals, either caspase-8 or -9 is capable of activating effector caspases such as caspase-3 or -7, which then cleave apoptotic substrates, leading to apoptosis. A link between the extrinsic and intrinsic pathways is observed in certain cells that involves the cleavage of the Bcl-2 family member Bid by caspase-8: this leads to release of cytochrome c from mitochondria and activation of caspase-9 (Muro, 2002 and references therein).

Certain IAP proteins have been shown to inhibit both apical and effector caspases, such as mammalian XIAP, where the third BIR domain of XIAP directly binds and inhibits caspase-9, whereas a short linker region between the first and second BIR domains binds and inhibits caspases 3 and 7. Inhibition of caspase-9 is relieved by Smac/DIABLO, another protein that is released from mitochondria following a death signal and that binds to BIR3 of XIAP, releasing caspase-9. The Drosophila DIAP1 protein is also capable of inhibiting death induced by ectopic caspase expression in yeast and in the fly eye (Hawkins, 2000; Meier, 2000; Wang, 1999). This activity is important for the anti-apoptotic function of DIAP1 because loss of DIAP1 results in caspase activation and the death of most, if not all, cells in the embryo (Wang, 1999; Goyal, 2000; Lisi, 2000; Yoo, 2002). In contrast, the xiap knockout mouse has no discernible phenotype, although the levels of c-IAP1 and c-IAP2 are higher than normal in embryonic fibroblasts derived from XIAP-deficient mice, suggesting compensation because of the loss of XIAP (Harlin, 2000; Muro, 2002 and references therein).

In Drosophila, a pathway similar to the intrinsic pathway in mammals is beginning to be characterized. A protein with homology to Apaf-1, known variously as DARK, Hac-1, or Dapaf-1, has been shown to be important for apoptosis stimulated by a variety of signals. In addition, the Drosophila caspases dronc/Nedd2-like caspase and Ice have been shown to accumulate in large complexes reminiscent of apoptosomes (Dorstyn, 2002). However, cytochrome c release does not appear to occur in Drosophila cells, and the role of cytochrome c in Drosophila apoptosome formation is not clear (Muro, 2002 and references therein).

Although loss of Thread has been shown to result in caspase activation and spontaneous cell death in Drosophila cells and embryos, the point at which DIAP1 normally functions to inhibit caspase activation has been uncertain. Depletion by RNA interference (RNAi) or cycloheximide treatment of the DIAP1 protein in Drosophila S2 cells results in rapid and widespread caspase-dependent apoptosis. Co-silencing of dronc, a gene coding for one of the apical caspases of Drosophila, or of dark, a pro-apoptotic regulatory protein, largely suppresses this apoptosis, indicating that DIAP1 is normally required to inhibit an activity dependent on these proteins. Silencing of dronc also inhibits processing of the effector caspase Ice following stimulation of apoptosis, demonstrating that DRONC functions as an apical caspase in S2 cells. Silencing of diap1 or treatment with UV light induces DRONC processing, which occurs in two steps. The first step appears to occur continuously even in the absence of an apoptotic signal and to be dependent on DARK, because full-length DRONC accumulates when dark is silenced in non-apoptotic cells. In addition, treatment with the proteasome inhibitor MG132 results in accumulation of this initially processed form of DRONC, but not full-length DRONC, in non-apoptotic cells. The second step in DRONC processing is observed only in apoptotic cells. These results indicate that the initial step in DRONC processing occurs continuously via a DARK-dependent mechanism in Drosophila cells and that DIAP1 is required to prevent excess accumulation of this first form of processed DRONC, presumably through its ability to act as a ubiquitin-protein ligase (Muro, 2002).

Based on these and other results, it has been hypothesized that a low level of constitutive caspase activity is present in cells, and IAP proteins promote survival by suppressing amplification of the caspase cascade. Disruption of IAP-caspase interactions thus provides an attractive approach to sensitizing cells to death signals. However, an important unanswered question remains: which IAP-caspase interactions are rate-limiting for the survival of cells that normally live? The RNAi technique has been used to dissect this pathway in insect cells. The results demonstrate that in unstimulated Drosophila S2 cells, DIAP1 is required to inhibit an activity dependent on DRONC and DARK. Furthermore, DRONC is continuously processed in normal cells through a mechanism that requires DARK. However, this initially processed form of DRONC does not normally accumulate to significant levels because it is subject to degradation by the proteasome. Removal of DIAP1 results in the accumulation of processed DRONC, activation of the downstream caspase Ice, and apoptosis (Muro, 2002).

GENE STRUCTURE

cDNA clone length - 2015

Bases in 5' UTR - 430

Exons - 2

Bases in 3' UTR - 267

PROTEIN STRUCTURE

Amino Acids - 438

Structural Domains

The inhibitor of apoptosis (IAP) proteins are found in all animals and regulate apoptosis (programmed cell death) by binding and inhibiting caspase proteases. This inhibition is overcome by several apoptosis stimulators, including Drosophila Hid and mammalian Smac/DIABLO, which bind to 65-residue baculovirus IAP repeat (BIR) domains found in one to three copies in all IAPs. Virtually all BIRs contain three Cys and a His that bind zinc, a Gly in a tight turn, and an Arg. The functional and structural role of the Arg has been investigated in isolated BIR domains from the baculovirus Orgyia pseudotsugata Op-IAP and the Drosophila DIAP1 proteins. Mutation of the Arg to either Ala or Lys abolishes Hid and Smac binding to BIRs, despite the Hid/Smac binding site being located on the opposite side of the BIR domain from the Arg. The mutant BIR domains also exhibit weakened zinc binding, increased sensitivity to limited proteolysis, and altered circular dichroism spectra indicative of perturbed domain folding. Examination of known BIR structures indicates that the Arg side chain makes simultaneous bridging hydrogen bonds and a cation-pi interaction for which the Arg guanidino group is uniquely well suited. These interactions are likely critical for stabilizing the tertiary fold of BIR domains in all IAPs, explaining the conservation of this residue (Luque, 2002).

date revised: 11 August 2003

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