The Drosophila Inhibitor of Apoptosis (IAP) DIAP2 Is Dispensable for Cell Survival, Required for the Innate Immune Response to Gram-negative Bacterial Infection, and Can Be Negatively Regulated by the Reaper/Hid/Grim Family of IAP-binding Apoptosis Inducers

Abstract

Many inhibitor of apoptosis (IAP) family proteins inhibit apoptosis. IAPs contain N-terminal baculovirus IAP repeatdomains and a C-terminal RING ubiquitin ligase domain. Drosophila IAP DIAP1 is essential for the survival of many cells, protecting them from apoptosis by inhibiting active caspases. Apoptosis initiates when proteins such as Reaper, Hid, and Grim bind a surface groove in DIAP1 baculovirus IAP repeat domains via an N-terminal IAP-binding motif. This evolutionarily conserved interaction disrupts DIAP1-caspase interactions, unleashing apoptosis-inducing caspase activity. A second Drosophila IAP, DIAP2, also binds Rpr and Hid and inhibits apoptosis in multiple contexts when overexpressed. However, due to a lack of mutants, little is known about the normal functions of DIAP2. We report the generation of diap2 null mutants. These flies are viable and show no defects in developmental or stress-induced apoptosis. Instead, DIAP2 is required for the innate immune response to Gram-negative bacterial infection. DIAP2 promotes cytoplasmic cleavage and nuclear translocation of the NF-B homolog Relish, and this requires the DIAP2 RING domain. Increasing the genetic dose of diap2 results in an increased immune response, whereas expression of Rpr or Hid results in down-regulation of DIAP2 protein levels. Together these observations suggest that DIAP2 can regulate immune signaling in a dose-dependent manner, and this can be regulated by IBM-containing proteins. Therefore, diap2 may identify a point of convergence between apoptosis and immune signaling pathways. The inhibitor of apoptosis (IAP)2 family proteins contain one or more repeats of an ϳ70-amino acid motif known as a baculovirus IAP repeat (BIR), which mediates interactions with multiple death activators and plays an essential role in the ability of these proteins to inhibit cell death. IAPs also contain a C-terminal RING E3 ubiquitin ligase domain that can target bound proteins, as well as the IAP itself, for ubiquitination and in some cases degradation (1). The Drosophila genome encodes two BIR and RING domain-containing IAP family members, DIAP1 and DIAP2, and ectopic expression of either protein inhibits apoptosis (2-4). DIAP1 is required continuously in many cells to inhibit the apical caspase Dronc and effector caspases activated by Dronc, such as Drice (5, 6). Critical interactions between caspases and DIAP1 are mediated by a surface groove within each DIAP1 BIR domain and short IAP-binding motifs (IBM) present in Dronc or Drice (7-9). Apoptosis in the fly can be induced by expression of proteins such as Reaper, Hid, Grim, Sickle, and Jafrac2 (the RHG proteins). Each of these proteins contains an N-terminal IBM that mediates competitive binding to DIAP1 through the same BIR surface grooves that are required for DIAP1-caspase interactions (8, 10). RHG proteins can also promote ubiquitin-dependent degradation of DIAP1. Both activities have the effect of liberating active caspases, resulting in apoptosis (6). IAPs that inhibit apoptosis, as well as inhibi...

Inhibitor of Apoptosis (IAP) Proteins-Modulators of Cell Death and Inflammation

“…NF-kB transcription factors are important regulators of the genes necessary for innate and adaptive immune responses and for the survival and proliferation of certain cell types (Karin and Greten 2005). The realization that IAPs function as critical components of NF-kB signal transduction first came from Drosophila where DIAP2 was found to be essential to fend off Gram-negative bacterial infection (Gesellchen et al 2005;Kleino et al 2005;Leulier et al 2006a;Huh et al 2007). In Drosophila, infection by Gram-negative bacteria triggers the innate immune response by activating the immune deficiency (IMD) signaling cascade (Lemaitre and Hoffmann 2007), a Rel/NF-kB-dependent pathway that shares striking similarities with mammalian tumor necrosis factor receptor 1 (TNF-R1) (Tanji and Ip 2005).…”

“…In Drosophila, infection by Gram-negative bacteria triggers the innate immune response by activating the immune deficiency (IMD) signaling cascade (Lemaitre and Hoffmann 2007), a Rel/NF-kB-dependent pathway that shares striking similarities with mammalian tumor necrosis factor receptor 1 (TNF-R1) (Tanji and Ip 2005). diap2 mutant flies fail to activate NF-kB-mediated expression of antibacterial peptide genes and, consequently, rapidly succumb to bacterial infection (Leulier et al 2006a;Huh et al 2007). DIAP2-mediated signaling to NF-kB critically depends on its Ub-E3 ligase activity (Leulier et al 2006a;Huh et al 2007;Paquette et al 2010;Meinander et al 2012).…”

“…diap2 mutant flies fail to activate NF-kB-mediated expression of antibacterial peptide genes and, consequently, rapidly succumb to bacterial infection (Leulier et al 2006a;Huh et al 2007). DIAP2-mediated signaling to NF-kB critically depends on its Ub-E3 ligase activity (Leulier et al 2006a;Huh et al 2007;Paquette et al 2010;Meinander et al 2012). In conjunction with the E2 Ub-conjugating enzymes Effete (UBC5) and UEV1a/ Bendless (UEV1a/Ubc13) (Zhou et al 2005;Paquette et al 2010), DIAP2 promotes the conjugation of K63-linked Ub chains on IMD and DREDD (also referred to as DCP-2) (Paquette et al 2010;Meinander et al 2012), the Drosophila ortholog of caspase-8.…”

Selection of <I>D2S3</I>, an <I>Aedes aegypti</I> (Diptera: Culicidae) Strain with High Oral Susceptibility to Dengue 2 Virus and <I>D2MEB</I>, a Strain with a Midgut Barrier to Dengue 2 Escape

“…Inhibitors of apoptosis impede activation of initiator and executioner caspases preventing either their dimerization or their binding to the active catalytic site of these enzymes (Huh et al 2007;Leu et al 2007). Some inhibitors of apoptosis have been identified and characterized in insects, but their significance during arbovirus infection in mosquitoes has not been completely elucidated (Blitvich et al 2002;Li et al 2007;Bryant et al 2008).…”

Ubr3 E3 ligase regulates apoptosis by controlling the activity of DIAP1 in Drosophila

“…The Drosophila genome encodes four IAPs, including Drosophila inhibitor of apoptosis protein 1 (DIAP1), DIAP2, DBruce and Deterin. [7][8][9][10] Among these four proteins, DIAP1 is stringently required to prevent caspase activation. 11,12 Although the requirement of DIAP1 in the apoptosis pathway is well documented, it is unclear how the activity of DIAP1 is regulated during development.…”