Apoptosis in Drosophila
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Apoptosis in Drosophila
Apoptosis refers to an evolutionarily conserved method of Cell Death that is characterized by specific morphological and biochemical properties.  Morphologically, Apoptosis is characterized by a series of structural changes in dying cells: Blebbing of the plasma membrane, Condensation of the cytoplasm and nucleus, and Cellular Fragmentation into membrane Apoptotic bodies. Biochemically, Apoptosis is characterized by the degradation of Chromatin, initially into large fragments of 50-300 kilobases and subsequently into smaller fragments that are monomers and multimers of 200 bases. Programmed Cell Death by Apoptosis is a common way for deleting unwanted and superfluous cells. It is crucial during development as well as in the maintenance of tissue homeostasis and regulation of certain diseases (Ref.1). Apoptosis in Drosophila involves three death-inducing genes, HID (Head Involution Defective), Rpr (Reaper), and Grim, which are required for essentially all normally occurring cell deaths in Drosophila embryogenesis. Central to the process is the gene Rpr. Rpr is a small protein of 65 amino acids and possesses a conserved sequence domain called the "Death Domain”. Two other Drosophila genes, Grim and HID, are closely linked to Rpr on the chomosome. Both proteins bear sequence homology to Rpr and other Death Domain proteins and both are involved in Programmed Cell Death. HID is a large novel 410 amino acid protein. The Grim gene product function in a parallel circuit of Cell death signaling that ultimately activates a common set of downstream Apoptotic effectors. More recently, an additional family member called Skl (Sickle) has been identified in flies (Ref.2).

Activities of these Apoptotic regulators are governed by diverse Developmental and Environmental signals. The expression and activity of HID are negatively regulated by the Ras1/ MAPK (Mitogen-Activated Protein Kinase) pathway, while Rpr expression is directly regulated by the p53/DNA damage pathway and the EcR (Ecdysone Receptor) signaling cascade. The Ras1 pathway inhibits HID activity apparently by the direct phosphorylation of HID by MAPK, Rl (Rolled). Alteration of Rl phosphorylation sites within the HID sequence blocks the survival signals generated by Rl. Rl is activated by Raf1, which is in turn, activated by Ras1. Ras1 is a membrane-associated Guanine nucleotide-Binding Protein that is normally activated in response to the binding of extracellular signals, such as Growth Factors, to RTKs (Receptor Tyrosine Kinases). These receptors include the dEGFR (Drosophila Epidermal Growth Factor Receptor), Sevenless and Torso. Extracellular signals enhance binding of dEGFRL (Drosophila Epidermal Growth Factor Receptor Ligand) to dEGFR. In flies, cells undergoing Apoptosis in response to DNA damage requires the activity of the Transcription factor p53. p53 can directly bind to a radiation-inducible enhancer in the Rpr promoter. p53 induces Apoptosis by activating Rpr expression. Drosophila TNF (Tumor Necrosis Factor) ligand, known as Eiger also triggers Cell Death. Eiger stimulates cell death through Caspase-dependent and Caspase-independent mechanisms and the latter rely on activation of the JNK (Jun N-terminal kinase) pathway. Eiger interacts physically with its receptor Wgn (Wengen) through its TNFR homology domain and TNF homology domain, respectively. Many other developmentally important pathways also regulate the tissue-specific expression of these genes. The Ban (Bantam) gene of Drosophila stimulates cell proliferation and prevents Apoptosis. The pro-apoptotic gene HID has been identified as a target for regulation by Ban miRNA (Micro-RNA), providing an explanation for Bans anti-apoptotic activity (Ref.3).

Each of the three Apoptotic genes, HID, Rpr, and Grim is sufficient to induce Apoptosis in a Caspase-dependent manner in Drosophila. These genes control Caspase activation by multiple mechanisms, including formation of an Apoptosome-Like complex containing DARK (Drosophila Apaf-1-Related Killer) and DARK-independent activation of downstream Caspases through antagonizing Caspase inhibitors such as DIAP1 (Drosophila Inhibitor of Apoptosis-1). They further impact Caspase activation by regulating conformational change or release of CytC (Cytochrome-C). There are a total of seven Caspases in Drosophila melanogaster: DCp1 (Death Caspase-1), Dredd (Death related ced-3/Nedd2-like protein), Drice, Dronc, DECAY (Death Executioner Caspase related to Apopain/Yama), DAMM (Death Associated Molecule related to Mch2), and Strica. Dredd and Dronc contain long prodomains carrying DEDs (Death Effector Domains) and a CARD (Caspase Recruitment Domains), respectively, suggesting that these two Caspases may act as upstream Caspases. Five Drosophila Caspases lack any homotypic motifs in their N-terminal regions and thus are likely to be downstream effector Caspases. Two of these, Drice and DCp1, are the most abundant and share the most sequence similarity to each other and to mammalian Caspase3. As in mammals, Drice and DCp1 are targets for Dronc-mediated processing and activation; however, activation of effector Caspases may also be independent of Dronc in certain contexts (Ref.4). Rpr, Grim, and HID triggers Dredd protein precursor processing by activating APAF1 (Apoptotic Protease Activating Factor-1) homologue ARK (APAF1-Related-Killer)/DARK/Dapaf1/Hac1. The long form of DARK most closely resembles APAF1, in that it contains a series of COOH-terminal WD-40 repeats that presumably mediate regulation by CytC. The short form most closely resembles Ced4, which lacks these repeats, and would thus be predicted to be constitutively active. Dredd and Dronc associate with DARK to activate the effector Caspases. Interactions between DARK, CytC, and Drosophila Caspases lead to Apoptosis. Mitochondrial CytC is shifted in localization, and released into the cytoplasm during Apoptosis. In Drosophila, CytC functions to transduce Apoptotic signals through APAF1. Down-regulation of DARK expression in Drosophila cells by dsRNA (Double stranded Ribonucleic Acid) resulted in complete resistance to UV (Ultra Violet)-, Cycloheximide-, and Actinomycin-D induced Apoptosis. However, removal of DARK did not prevent Rpr or Grim induced Apoptosis in Drosophila cells (Ref.5).

In addition to three positive effectors of Apoptosis in Drosophila, two negative regulators, DIAP1 (Drosophila Inhibitor of Apoptosis-1) and DIAP2 (Drosophila Inhibitor of Apoptosis-2), have also been found. Rpr, HID, and Grim share a short region of homology at the N-terminus that is capable of interacting with an inhibitor of Apoptosis protein, DIAP1. This N-terminal region, termed an IBM (IAP-Binding Motif) or RHG (Reaper-HID-Grim) domain, is conserved among other IAPs (IAP-Binding Proteins) in flies. The IBM proteins promote Apoptosis by binding to and antagonizing IAPs, thereby liberating IAPs from inhibiting Caspases. Inhibition of DIAP1, either via expression of Rpr and HID or by mutational inactivation, led to the induction of the putative mitogens Wgn (Wengen) and Dpp (Decapentaplegic). In addition to their IAP binding function, Rpr and Grim may also promote Apoptosis by generally inhibiting translation (Ref.6).

Bcl2 (B-Cell CLL/Lymphoma-2) family also regulates Apoptosis in Drosophila. In Drosophila, there are two homologs of the Bcl2/Ced9 family of PCD (Programmed Cell Death) proteins, DEBCL (Death Executioner Bcl2 homologue)/dBorg1/dRob1 and Buffy/dBorg-2. Although both DEBCL and Buffy share the BH1, BH2, BH3 and C-terminal transmembrane domains of the Bcl2 family of proteins, they appear to lack the N-terminal BH4 domain.  DEBCL has been shown to have a pro-apoptotic function in Drosophila, whereas Buffy is required for cell survival and can prevent developmental and irradiation-induced cell death. Buffy interacts genetically and physically with DEBCL to suppress DEBCL-induced cell death.  In the fly, increased levels of Buffy are sufficient to inhibit the Drosophila Apoptotic pathway that normally responds to DNA damaging agents. Anti- and proapoptotic Bcl2 family homologs in Drosophila reside downstream or in parallel to RHG proteins and further influence Caspase activation. The miRNA encoding gene Mir14 impact Apoptosis in flies by suppressing Drice. Drosophila is an appropriate animal model for the genetic analysis of Apoptosis and also has emerged as a model for neurodegenerative diseases such as Huntingtons disease and Machado-Joseph disease (Ref.7 and Ref.8).