Diap1: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - Death-associated inhibitor of apoptosis 1

Synonyms - DIAP1, thread

Cytological map position - 72D1

Function - signaling

Keywords - apoptosis, protein degradation, E3 ubiquitin ligase

Symbol - Diap1

FlyBase ID: FBgn0260635

Genetic map position - 3-43.2

Classification - Inhibitor of apoptosis (IAP) repeat

Cellular location - cytoplasmic

NCBI link: Entrez Gene
Diap1 orthologs: Biolitmine
Recent literature
Hasan, S., Hétié, P. and Matunis, E.L. (2015). Niche signaling promotes stem cell survival in the Drosophila testis via the Jak-STAT target DIAP1. Dev Biol [Epub ahead of print]. PubMed ID: 25941003
Tissue-specific stem cells are thought to resist environmental insults better than their differentiating progeny, but this resistance varies from one tissue to another, and the underlying mechanisms are not well-understood. This study uses the Drosophila testis as a model system to study the regulation of cell death within an intact niche. This niche contains sperm-producing germline stem cells (GSCs) and accompanying somatic cyst stem cells (or CySCs). Although many signals are known to promote stem cell self-renewal in this tissue, including the highly conserved JAK-STAT pathway, the response of these stem cells to potential death-inducing signals, and factors promoting stem cell survival, have not been characterized. The study found that both GSCs and CySCs resisted cell death better than their differentiating progeny, under normal laboratory conditions and in response to potential death-inducing stimuli such as irradiation or starvation. To ask what might be promoting stem cell survival, the study characterized the role of the anti-apoptotic gene Drosophila inhibitor of apoptosis 1 (diap1) in testis stem cells. DIAP1 protein was enriched in the GSCs and CySCs and was a Jak-STAT target. diap1 was necessary for survival of both GSCs and CySCs, and ectopic up-regulation of DIAP1 in somatic cyst cells was sufficient to non-autonomously rescue stress-induced cell death in adjacent differentiating germ cells (spermatogonia). Altogether, these results show that niche signals can promote stem cell survival by up-regulation of highly conserved anti-apoptotic proteins, and suggest that this strategy may underlie the ability of stem cells to resist death more generally.

Pushpavalli, S. N., Sarkar, A., Ramaiah, M. J., Koteswara Rao, G., Bag, I., Bhadra, U. and Pal-Bhadra, M. (2015). Drosophila MOF regulates DIAP1 and induces apoptosis in a JNK dependent pathway. Apoptosis [Epub ahead of print]. PubMed ID: 26711898
Histone modulations have been implicated in various cellular and developmental processes where in Drosophila Mof is involved in acetylation of H4K16. Reduction in the size of larval imaginal discs is observed in the null mutants of mof with increased apoptosis. Deficiency involving Hid, Reaper and Grim [H99] alleviated mof RNAi induced apoptosis in the eye discs. mof RNAi induced apoptosis leads to activation of caspases which is suppressed by over expression of caspase inhibitors like P35 and Diap1 clearly depicting the role of caspases in programmed cell death. Also apoptosis induced by knockdown of mof is rescued by JNK mutants of bsk and tak1 indicating the role of JNK in mof RNAi induced apoptosis. The adult eye ablation phenotype produced by ectopic expression of Hid, Rpr and Grim, was restored by over expression of Mof. Accumulation of Mof at the Diap1 promoter 800 bp upstream of the transcription start site in wild type larvae is significantly higher (up to twofolds) compared to mof mutants. This enrichment coincides with modification of histone H4K16Ac indicating an induction of direct transcriptional up regulation of Diap1 by Mof. Based on these results it is proposed that apoptosis triggered by mof RNAi proceeds through a caspase-dependent and JNK mediated pathway.

Hwangbo, D. S., Biteau, B., Rath, S., Kim, J. and Jasper, H. (2016). Control of apoptosis by Drosophila DCAF12. Dev Biol [Epub ahead of print]. PubMed ID: 26972874
Regulated Apoptosis (Programmed Cell Death, PCD) maintains tissue homeostasis in adults, and ensures proper growth and morphogenesis of tissues during development of metazoans. Accordingly, defects in cellular processes triggering or executing apoptotic programs have been implicated in a variety of degenerative and neoplastic diseases. This study reports the identification of DCAF12, an evolutionary conserved member of the WD40-motif repeat family of proteins, as a new regulator of apoptosis in Drosophila. DCAF12 is required for Diap1 cleavage in response to pro-apoptotic signals, and is thus necessary and sufficient for RHG (Reaper, Hid, and Grim)-mediated apoptosis. Loss of DCAF12 perturbs the elimination of supernumerary or proliferation-impaired cells during development, and enhances tumor growth induced by loss of neoplastic tumor suppressors, highlighting the wide requirement for DCAF12 in PCD.
Wang, Z., Xia, X., Yang, X., Zhang, X., Liu, Y., Wu, D., Fang, Y., Liu, Y., Xu, J., Qiu, Y. and Zhou, X. (2017). A picorna-like virus suppresses the N-end rule pathway to inhibit apoptosis. Elife 6. PubMed ID: 29231806
The N-end rule pathway is an evolutionarily conserved proteolytic system that degrades proteins containing N-terminal degradation signals called N-degrons, and has emerged as a key regulator of various processes. Viruses manipulate diverse host pathways to facilitate viral replication and evade antiviral defenses. However, it remains unclear if viral infection has any impact on the N-end rule pathway. In this study, using a picorna-like virus as a model, it was found that viral infection promoted the accumulation of caspase-cleaved Drosophila inhibitor of apoptosis 1 (DIAP1) by inducing the degradation of N-terminal amidohydrolase 1 (NTAN1), a key N-end rule component that identifies N-degron to initiate the process. The virus-induced NTAN1 degradation is independent of polyubiquitylation but dependent on proteasome. Furthermore, the virus-induced N-end rule pathway suppression inhibits apoptosis and benefits viral replication. Thus, these findings demonstrate that a virus can suppress the N-end rule pathway, and uncover a new mechanism for virus to evade apoptosis.
Wang, Z., Xia, X., Yang, X., Zhang, X., Liu, Y., Wu, D., Fang, Y., Liu, Y., Xu, J., Qiu, Y. and Zhou, X. (2017). A picorna-like virus suppresses the N-end rule pathway to inhibit apoptosis. Elife 6. PubMed ID: 29231806
The N-end rule pathway is an evolutionarily conserved proteolytic system that degrades proteins containing N-terminal degradation signals called N-degrons, and has emerged as a key regulator of various processes. Viruses manipulate diverse host pathways to facilitate viral replication and evade antiviral defenses. However, it remains unclear if viral infection has any impact on the N-end rule pathway. In this study, using a picorna-like virus as a model, it was found that viral infection promoted the accumulation of caspase-cleaved Drosophila inhibitor of apoptosis 1 (DIAP1) by inducing the degradation of N-terminal amidohydrolase 1 (NTAN1), a key N-end rule component that identifies N-degron to initiate the process. The virus-induced NTAN1 degradation is independent of polyubiquitylation but dependent on proteasome. Furthermore, the virus-induced N-end rule pathway suppression inhibits apoptosis and benefits viral replication. Thus, these findings demonstrate that a virus can suppress the N-end rule pathway, and uncover a new mechanism for virus to evade apoptosis.
Reiff, T., Antonello, Z. A., Ballesta-Illan, E., Mira, L., Sala, S., Navarro, M., Martinez, L. M. and Dominguez, M. (2019). Notch and EGFR regulate apoptosis in progenitor cells to ensure gut homeostasis in Drosophila. EMBO J: e101346. PubMed ID: 31566767 The regenerative activity of adult stem cells carries a risk of cancer, particularly in highly renewable tissues. Members of the family of inhibitor of apoptosis proteins (IAPs) inhibit caspases and cell death, and are often deregulated in adult cancers; however, their roles in normal adult tissue homeostasis are unclear. This study show that regulation of the number of enterocyte-committed progenitor (enteroblast) cells in the adult Drosophila involves a caspase-mediated physiological apoptosis, which adaptively eliminates excess enteroblast cells produced by intestinal stem cells (ISCs) and, when blocked, can also lead to tumorigenesis. Importantly, it was found that Diap1 is expressed by enteroblast cells and that loss and gain of Diap1 led to changes in enteroblast numbers. Antagonistic interplay between Notch and EGFR signalling was found to govern enteroblast life/death decisions via the Klumpfuss/WT1 and Lozenge/RUNX transcription regulators, which also regulate enteroblast differentiation and cell fate plasticity. These data provide new insights into how caspases drive adult tissue renewal and protect against the formation of tumours.
Kidera, H., Hatabu, T. and Takahashi, K. H. (2020). Apoptosis inhibition mitigates aging effects in Drosophila melanogaster. Genetica. PubMed ID: 32219590
Aging is a natural biological process that results in progressive loss of cell, tissue, and organ function. One of the causing factors of the aging process is the decrease in muscle mass, which has not been fully verified in Drosophila. Apoptotic cell death may result in aberrant cell loss and can eventually diminish tissue function and muscle atrophy. If so, inhibition of apoptosis may prolong longevity and reduce motor function and muscle mass decline with age in Drosophila flies. This study used Drosophila melanogaster as study material, and induced the overexpression of Drosophila inhibitor of apoptosis protein 1 gene to inhibit apoptosis, and investigated the effect of apoptosis inhibition on the longevity and age-related declines in flight and climbing ability and muscle mass. As a result, the inhibition of apoptosis tended to mitigate the aging effects and prolonged longevity and reduced climbing ability decline with age. The current study suggests that apoptosis inhibition could mitigate the aging effects in D. melanogaster. Although such effects have already been known in mammals, the current results suggest that the apoptosis may play a similar role in insects as well.
Ahmad, V., Vadla, G. P. and Chabu, C. Y. (2020). Syd/JIP3 controls tissue size by regulating Diap1 protein turnover downstream of Yorkie/YAP. Dev Biol 469: 37-45. PubMed ID: 33022230
How organisms control organ size is not fully understood. This study found that Syd/JIP3 is required for proper wing size in Drosophila. JIP3 mutations are associated with organ size defects in mammals. The underlying mechanisms are not well understood. Syd/JIP3 inhibition was found to result in a downregulation of the inhibitor of apoptosis protein 1 (Diap1) in the Drosophila wing. Correspondingly, Syd/JIP3 deficient tissues exhibit ectopic cell death and yield smaller wings. Syd/JIP3 inhibition generated similar effects in mammalian cells, indicating a conserved mechanism. Yorkie/YAP stimulates Syd/JIP3 in Drosophila and mammalian cells. Notably, Syd/JIP3 is required for the full effect of Yorkie-mediated tissue growth. Thus Syd/JIP3 regulation of Diap1 functions downstream of Yorkie/YAP to control growth. This study provides mechanistic insights into the recent and perplexing link between JIP3 mutations and organ size defects in mammals, including in humans where de novo JIP3 variants are associated with microcephaly.
Kassel, S., Hanson, A. J., Benchabane, H., Saito-Diaz, K., Cabel, C. R., Goldsmith, L., Taha, M., Kanuganti, A., Ng, V. H., Xu, G., Ye, F., Picker, J., Port, F., Boutros, M., Weiss, V. L., Robbins, D. J., Thorne, C. A., Ahmed, Y. and Lee, E. (2023). USP47 deubiquitylates Groucho/TLE to promote Wnt-β-catenin signaling. Sci Signal 16(771): eabn8372. PubMed ID: 36749823
The Wnt-β-catenin signal transduction pathway is essential for embryonic development and adult tissue homeostasis. Wnt signaling converts TCF from a transcriptional repressor to an activator in a process facilitated by the E3 ligase XIAP. XIAP-mediated monoubiquitylation of the transcriptional corepressor Groucho (also known as TLE) decreases its affinity for TCF, thereby allowing the transcriptional coactivator β-catenin to displace it on TCF. Through a genome-scale screen in cultured Drosophila melanogaster cells, this study identified the deubiquitylase USP47 as a positive regulator of Wnt signaling. USP47 was found to be required for Wnt signaling during Drosophila and Xenopus laevis development, as well as in human cells, indicating evolutionary conservation. In human cells, knockdown of USP47 inhibited Wnt reporter activity, and USP47 acted downstream of the β-catenin destruction complex. USP47 interacted with TLE3 and XIAP but did not alter their amounts; however, knockdown of USP47 enhanced XIAP-mediated ubiquitylation of TLE3. USP47 inhibited ubiquitylation of TLE3 by XIAP in vitro in a dose-dependent manner, suggesting that USP47 is the deubiquitylase that counteracts the E3 ligase activity of XIAP on TLE. These data suggest a mechanism by which regulated ubiquitylation and deubiquitylation of TLE enhance the ability of β-catenin to cycle on and off TCF, thereby helping to ensure that the expression of Wnt target genes continues only as long as the upstream signal is present.
Maurya, C. K. and Tapadia, M. G. (2023). Expanded polyQ aggregates interact with sarco-endoplasmic reticulum calcium ATPase and Drosophila inhibitor of apoptosis protein1 to regulate polyQ mediated neurodegeneration in Drosophila. Mol Cell Neurosci 126: 103886. PubMed ID: 37567489
Polyglutamine (polyQ) induced neurodegeneration is one of the leading causes of progressive neurodegenerative disorders characterized clinically by deteriorating movement defects, psychiatric disability, and dementia. Calcium [Ca(2+)] homeostasis, which is essential for the functioning of neuronal cells, is disrupted under these pathological conditions. In this paper, we simulated Huntington's disease phenotype in the neuronal cells of the Drosophila eye, and [Ca(2+)] pump, sarco-endoplasmic reticulum calcium ATPase (SERCA), was identified as one of the genetic modifiers of the neurodegenerative phenotype. This paper shows genetic and molecular interaction between polyglutamine (polyQ) aggregates, SERCA and DIAP1. Evidence is presented that polyQ aggregates interact with SERCA and alter its dynamics, resulting in a decrease in cytosolic [Ca(2+)] and an increase in ER [Ca(2+)], and thus toxicity. Downregulating SERCA lowers the enhanced calcium levels in the ER and rescues, morphological and functional defects caused due to expanded polyQ repeats. Cell proliferation markers such as Yorkie (Yki), Scalloped (Sd), and phosphatidylinositol 3 kinases/protein kinase B (PI3K/Akt), also respond to varying levels of calcium due to genetic manipulations, adding to the amelioration of degeneration. These results imply that neurodegeneration due to expanded polyQ repeats is sensitive to SERCA activity, and its manipulation can be an important step toward its therapeutic measures.
Park, J. E., Lee, J., Ok, S., Byun, S., Chang, E. J., Yoon, S. E., Kim, Y. J. and Kang, M. J. (2023). Wg/Wnt1 and Erasp link ER stress to proapoptotic signaling in an autosomal dominant retinitis pigmentosa model. Exp Mol Med 55(7): 1544-1555. PubMed ID: 37464094
The endoplasmic reticulum (ER) is a subcellular organelle essential for cellular homeostasis. Perturbation of ER functions due to various conditions can induce apoptosis. Chronic ER stress has been implicated in a wide range of diseases, including autosomal dominant retinitis pigmentosa (ADRP), which is characterized by age-dependent retinal degeneration caused by mutant rhodopsin alleles. However, the signaling pathways that mediate apoptosis in response to ER stress remain poorly understood. In this study, an unbiased in vivo RNAi screen was performed with a Drosophila ADRP model and found that Wg/Wnt1 mediated apoptosis. Subsequent transcriptome analysis revealed that ER stress-associated serine protease (Erasp), which has been predicted to show serine-type endopeptidase activity, was a downstream target of Wg/Wnt1 during ER stress. Furthermore, knocking down Erasp via RNAi suppressed apoptosis induced by mutant rhodopsin-1 (Rh-1(P37H)) toxicity, alleviating retinal degeneration in the Drosophila ADRP model. In contrast, overexpression of Erasp resulted in enhanced caspase activity in Drosophila S2 cells treated with apoptotic inducers and the stabilization of the initiator caspase Dronc (Death regulator Nedd2-like caspase) by stimulating DIAP1 (Drosophila inhibitor of apoptosis protein 1) degradation. These findings helped identify a novel cell death signaling pathway involved in retinal degeneration in an autosomal dominant retinitis pigmentosa model.

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).

Identification of E2/E3 ubiquitinating enzymes and caspase activity regulating Drosophila sensory neuron dendrite pruning

Ubiquitin-proteasome system (UPS) is a multistep protein degradation machinery implicated in many diseases. In the nervous system, UPS regulates remodeling and degradation of neuronal processes and is linked to Wallerian axonal degeneration, though the ubiquitin ligases that confer substrate specificity remain unknown. Having shown previously that class IV dendritic arborization (C4da) sensory neurons in Drosophila undergo UPS-mediated dendritic pruning during metamorphosis, an E2/E3 ubiquitinating enzyme mutant screen was conducted, revealing that mutation in ubcD1, an E2 ubiquitin-conjugating enzyme encoding Effete, resulted in retention of C4da neuron dendrites during metamorphosis. Further, UPS activation likely leads to UbcD1-mediated degradation of DIAP1, a caspase-antagonizing E3 ligase. This allows for local activation of the Dronc caspase, thereby preserving C4da neurons while severing their dendrites. Thus, in addition to uncovering E2/E3 ubiquitinating enzymes for dendrite pruning, this study provides a mechanistic link between UPS and the apoptotic machinery in regulating neuronal process remodeling (Kuo, 2006).

The ubiquitin-proteasome system (UPS), evolutionarily conserved for the regulation of protein turnover, targets proteins for degradation via a complex, temporally regulated process that results in proteasome-mediated destruction of polyubiquitinated proteins. There are two distinct steps involved: first, the covalent conjugation of ubiquitin polypeptide to the protein substrates, and second, the destruction of tagged proteins in the proteasome complex. The transfer of ubiquitin to a target molecule slated for degradation involves at least three enzymatic modifications: ubiquitin is first activated by the ubiquitin-activating enzyme E1; ubiquitin is then transferred to a carrier protein, a ubiquitin-conjugating enzyme E2, and finally, ubiquitin is transferred to a protein substrate bound by a ubiquitin ligase E3. There are minor variations to this enzymatic cascade, but overall, these highly specific protein-protein interactions ensure ubiquitin targeting specificity and regulate many aspects of housekeeping protein turnover and cellular maintenance. However, with the multiple regulatory layers, different parts of this complex machinery can break down. Mutations in the UPS pathway causing accumulation of nondegraded proteins have been implicated in a variety of human diseases (Kuo, 2006).

In the nervous system, aberrations in the UPS pathway have been implicated in disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and other neurodegenerative diseases. One of the common pathological features of neurodegenerative diseases, besides neuronal loss, is local axon degeneration. For example, in the case of Wallerian degeneration in vertebrates, distal parts of a severed axon remain viable and conduct action potentials in vivo for some time before a rapid dismantling of cytoskeletal proteins and axon degeneration, and the initiation of this rapid axon degeneration involves the UPS pathway. It is thought that UPS activation can lead to microtubule depolymerization and subsequent neurofilament degradation, possibly acting in conjunction with the Ca2+-dependent protease calpain. Moreover, inhibiting UPS activity in neurons prior to severing their axons can dramatically retard degradation of the severed axons. These results suggest that a cell-intrinsic UPS pathway regulates axon stability and that pharmaceutical inactivation of the UPS may prevent axonal degeneration in disease states (Kuo, 2006 and references therein).

In Drosophila, the remodeling of neuronal processes during normal development closely resembles the pathological phenotypes in Wallerian degeneration. In the mushroom body γ neurons, extensive pruning of larval axons occurs during metamorphosis in a process regulated by glia engulfment and neuron-intrinsic UPS activity. Similarly, in the fly peripheral nervous system, the class IV dendritic arborization (C4da) neurons undergo complete pruning of their extensive larval dendrites during metamorphosis, in a process that is also regulated by UPS activity (Kuo, 2005). In both of these examples, severing of neuronal processes is preceded by microtubule depolymerization and followed by cytoplasmic blebbing and degeneration, all phenotypes resembling Wallerian degeneration. Therefore, these fly neurons represent excellent systems in which to understand the roles of the UPS in regulating neuronal axon/dendrite integrity, given the rather limited knowledge of how the UPS participates in the degradation of neuronal processes. It is not known which specific E2 ubiquitin-conjugating enzyme(s) and E3 ubiquitin ligase(s) are involved in UPS-mediated remodeling/degradation of neuronal processes, or their specific downstream target(s) (Kuo, 2006).

It has been shown that mutations in the fly ubiquitin activation enzyme (uba1) and the proteasome complex (mov34) can prevent efficient pruning of C4da neuron larval dendrites during metamorphosis (Kuo, 2005). To further investigate the role of UPS in C4da neuron dendrite remodeling, a candidate gene screen was conducted to identify the E2 ubiquitin-conjugating enzyme and the E3 ubiquitin ligase required for this process. Analysis of genetic mutants showed that UPS activation in C4da neurons likely results in UbcD1 (an E2 ubiquitin-conjugating enzyme) mediated degradation of Drosophila inhibitor of apoptosis protein 1 (DIAP1), an E3 ligase that antagonizes caspase activity. Degradation of DIAP1 leads to activation of caspase Dronc, which results in local caspase activation and cleavage of proximal dendrites in C4da neurons during metamorphosis. In addition to the identification of a set of E2/E3 ubiquitinating enzymes for C4da neuron dendrite remodeling—with the surprising finding that the UPS mediates degradation of the potent protease inhibitor DIAP1—this study also establishes a mechanistic link between the UPS and caspase pathways in regulating C4da neuron dendrite pruning (Kuo, 2006).

To identify the E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase mediating dendrite pruning of C4da neurons during metamorphosis, a candidate gene approach was taken to systematically test the roles of known E2/E3 ubiquitinating enzymes in Drosophila. A set of putative E2/E3 ubiquitinating enzyme mutations was assembled, and live imaging was used to visualize C4da neurons carrying the E2/E3 mutation via the pickpocket(ppk)-EGFP marker, which specifically labels C4da neurons during Drosophila development. Those mutants with an early lethal phase were characterized by generating mosaic analysis with a repressible cell marker (MARCM) mutant neuronal clones. Since wild-type (wt) C4da neurons during metamorphosis do not retain any larval dendrites following head eversion, as imaged 18–20 hr after puparium formation (APF), mutations that caused larval dendrite retention in C4da neurons at this stage were sought. The candidate genes tested mostly showed no defects in dendrite pruning or neuronal cell death. However, one candidate, ubcD1, showed a modest level of larval dendrite retention at 18 hr APF (Kuo, 2006).

Live imaging of wt C4da neuron MARCM clones at the start of pupariation (white pupae, WP) showed primary and secondary dendritic branching patterns typical of C4da neurons. Consistent with previous reports (Kuo, 2005; Williams, 2005), wt C4da neurons sever their larval dendrites during early metamorphosis and by 18 hr APF are devoid of any dendrites. The ubcD1 mutant C4da MARCM clones showed similar dendritic morphology to the wt clones at the onset of metamorphosis. However, at 18 hr APF, the mutant clones consistently retained intact, nonsevered larval dendrites. Thus, the UbcD1 E2 ubiquitin-conjugating enzyme is required for proper UPS-mediated dendrite pruning in C4da neurons during metamorphosis (Kuo, 2006).

UbcD1, encoded by the gene effete, regulates UPS-mediated degradation of the antiapoptotic protein DIAP1 (Treier, 1992; Wang, 1999; Ryoo, 2002). In protecting cells from apoptosis, the DIAP1 E3 ubiquitin ligase antagonizes Dronc caspase activity by regulating ubiquination and degradation of the Dronc protein. Following apoptotic stimuli, UbcD1 mediates self-ubiquination and degradation of DIAP1, allowing for subsequent Dronc caspase activation. The biochemical and genetic interactions between these molecules are well established. The baculovirus p35, which is commonly used to inhibit caspase activity in Drosophila, and does not block C4da neuron dendrite pruning (Kuo, 2005). This may seem to make the involvement of caspases in this process unlikely; however, p35 has only limited activity against the caspase Dronc. To study the effects of dronc mutation on C4da neuron dendrite pruning, two null alleles of Dronc, dronc51 and dronc11, were used. MARCM analysis of dronc mutant clones revealed that the dendrites of mutant C4da neurons appeared normal at larval stages. However, unlike wt clones, without Dronc these neurons failed to properly prune their larval dendrites during metamorphosis, and most showed relatively intact primary and secondary larval dendritic arbors at 18 hr APF. These results show that severing of primary larval dendrites from C4da neurons during early metamorphosis requires the Dronc caspase (Kuo, 2006).

During apoptosis, Dronc activation requires the degradation of the antiapoptotic/anticaspase protein DIAP1, which is downstream of UbcD1. The requirement of UbcD1 for C4da neuron larval dendrite pruning during metamorphosis, together with the finding that Dronc caspase activity is also essential, raised the question of whether UPS-mediated DIAP1 degradation is a key step that allows for the severing of larval dendrites. Because loss of DIAP1 function causes C4da neuron cell death prior to the onset of metamorphosis, this question was approached using a gain-of-function allele of diap1, diap16-3s, which has a single amino acid mutation that makes DIAP1 an inefficient substrate for UPS-mediated degradation. ppk-EGFP was crossed into the gain-of-function mutant background and live imaging was used to follow C4da neuron dendrite pruning during metamorphosis. The diap16-3s mutation did not significantly affect the ability of C4da neurons to elaborate larval dendrites. However, unlike wt C4da neurons that completely pruned their larval dendrites by 18 hr APF, C4da neurons in the diap16-3s gain-of-function mutants failed to efficiently sever larval dendrites at 18 hr APF. These results suggest that the degradation of DIAP1 during early metamorphosis is required for proper C4da neuron larval dendrite pruning. Quantitatively, mutations in the UPS pathway that modulate Dronc activity (diap16-3s and ubcD1) resulted in less severe dendrite pruning defects than dronc mutants, both in terms of total number of large dendrites attached to soma and in the length of the longest attached dendrite at 18 hr APF (Kuo, 2006).

The UbcD1-DIAP1-Dronc pathway in apoptosis is well established. Thus, it may be necessary for C4da neurons to restrict the action of this pathway to specific cellular locations in order to prune unwanted dendrites without triggering apoptosis. To address this possibility, the subcellular distribution of DIAP1 and Dronc proteins was examined in ppk-EGFP C4da neurons. During the transition from third instar larvae to white pupae at the onset of metamorphosis, as well as 2 hr APF, there was a consistent induction of nuclear DIAP1 in GFP-labeled C4da neurons. During the same period a concurrent decrease was detected in Dronc staining in the soma of C4da neurons, unlike those from the neighboring cells at 2 hr APF. These results are consistent with previous observations that C4da neurons survive through this stage of metamorphosis. However, the level of antibody staining made it difficult to monitor the distribution of DIAP1 and Dronc within the dendritic structures of the C4da neurons. Because overexpression of Dronc caused C4da neuron to undergo apoptosis prior to metamorphosis, it was not possible to use GFP-tagged Dronc to examine its distribution in these neurons during pupariation. It was therefore necessary to search for alternative means to visualize activated Dronc or its downstream caspases (Kuo, 2006).

An antibody generated against activated mammalian caspase 3 has been shown to be effective in recognizing activated caspases in Drosophila. Whereas this antibody reportedly recognizes the Drosophila effector caspase Drice, it may also cross react with other activated Drosophila caspases such as Dronc during tissue staining, because of similarities in the sequences of these caspases in the region corresponding to the peptide used to generate this antibody. Therefore this antibody was used to determine whether activated caspase is localized to the dendrites of C4da neurons during the initial severing event. At 4 hr APF, just prior to dendrite severing, antibody staining for activated caspase was consistently observed within the proximal larval dendrites of C4da neurons, especially within dendritic swellings. In the diap16-3s gain-of-function mutant that inhibits Dronc activity, as well as in ubcD1 and dronc mutant MARCM clones, C4da neurons did not show dendritic swellings or activated caspase staining in dendrites during early metamorphosis. Consistent with previous observation that C4da neurons do not remodel their axons during concurrent dendrite pruning, no activated caspase staining was seen within the axons of C4da neurons during dendrite severing. Since overexpression of p35 in these neurons did not block dendrite pruning (Kuo, 2005), it is believed this antibody staining likely recognizes activated Dronc directly or recognizes a p35-resistant caspase that is activated by Dronc. These results show that, concurrent with the nuclear upregulation of DIAP1 in C4da neurons that prevents apoptosis, there is a local activation of caspases in the dendrites, likely as a result of UPS-mediated degradation of DIAP1. The spatially restricted activation of caspases then allows the severing of proximal larval dendrites from the soma (Kuo, 2006).

This study has shown that the UPS regulates pruning of larval dendrites from C4da neurons in a cell-intrinsic manner. To better understand the molecular pathways regulating UPS-mediated pruning, a candidate E2/E3 ubiquitinating enzyme screen was conducted. In this screen an E2 ubiquitin-conjugating enzyme mutation in was uncovered ubcD1, causing dendrite pruning defects. Taken together with the extensive biochemical characterization of interactions between UbcD1, DIAP1, and Dronc, this study suggests that in C4da neurons, UPS activation leads to UbcD1-mediated degradation of E3 ubiquitin ligase DIAP1, thereby allowing Dronc caspase activation and the subsequent cleavage of larval dendrites. This work not only identifies a set of E2/E3 ubiquitinating enzymes regulating neuronal process remodeling, it also links the UPS to a hitherto unappreciated mechanism for local caspase activation in dendrites during Drosophila metamorphosis (Kuo, 2006).

The mechanistic link between the UPS and caspase activity in regulating C4da neuron dendrite pruning is unexpected. Although the UPS is known to regulate remodeling and degradation of neuronal processes, it is generally believed that this process is accomplished by degradation of cellular proteins (such as microtubules and neurofilaments) that are required to keep dendrites and axons intact. However, it was found that the UPS in C4da neurons is in fact causing the degradation of an E3 ligase, DIAP1, thereby allowing for subsequent dendrite pruning. In this case, UPS-mediated degradation of a protein does not in and of itself lead to a structural compromise in dendrites, but rather it leads to the activation of another protease that executes dendrite pruning. This two-step activation cascade, which involves both the UPS and the apoptotic machinery, may provide an additional level of control and flexibility that would not be possible if UPS alone regulated the pruning program. After all, these C4da neurons, which are specified during fly embryogenesis, maintain a highly elaborate dendritic field to receive sensory inputs throughout larval development, which lasts for several days. The maintenance of these dendrites over time requires a network of finely tuned cell-intrinsic and -extrinsic pathways. Just as important, the dendritic pruning program enables dramatic neuronal remodeling in response to profound environmental changes during metamorphosis. It is conceivable that C4da neurons evolved this dual control mechanism to prevent any accidental triggering of dendrite pruning prior to metamorphosis. Initiation of C4da neuron dendrite pruning requires cell-intrinsic ecdysone signaling, and ecdysone receptors have been shown to regulate Dronc expression. It will be of interest to determine how this UPS/caspase dendritic pruning pathway is related to the ecdysone signaling cascade (Kuo, 2006).

During metamorphosis, C4da neurons upregulate DIAP1 expression in the nucleus, which is consistent with this class of neurons surviving early stages of the metamorphosis (only one of the three C4da neurons per hemisegment, the ventral neuron, is lost at a later stage of pupariation). Remarkably, there are activated caspases within the dendrites prior to severing, and a gain-of-function diap1 mutation can block dendrite pruning, strongly implicating a local dendritic program that can activate caspases without causing apoptosis of the neuron. Although mutations in both the Dronc caspase and the UPS pathway that modulate Dronc activity (UbcD1 and DIAP1) result in retention of larval dendrites, their dendrite pruning defects differed somewhat quantitatively. Compared to dronc mutants, diap1 gain-of-function and especially ubcD1 mutants showed less retention of larval dendrites during metamorphosis. This is not surprising for diap1 gain-of-function, as it is an effective Dronc inhibitor but unlikely to be 100% efficient. UbcD1, as an E2 ubiquitin-conjugating enzyme, has wider substrate specificity than E3 ligases. Previous study showed that UbcD1 is involved in mushroom body neuroblast proliferation, so it may be involved in other UPS-mediated pathways during dendrite pruning. It is also conceivable that in the absence of UbcD1 another E2 may trigger a low level of DIAP1 degradation, allowing residual Dronc activation which results in a milder dendrite pruning phenotype in ubcD1 mutants. It is currently unclear whether UbcD1 is also required during DIAP1-mediated degradation of Dronc. However, pruning defects in the ubcD1 mutants suggest that it may not be absolutely required, since undegraded DIAP1 continues to inhibit Dronc, presumably via interaction with another E2 protein (Kuo, 2006).

How is the specificity of dendrite pruning achieved? Several possible mechanisms are proposed: first, C4da neurons do not change their axonal projections during dendrite pruning, so there could be dendrite-specific trafficking of components of the UPS, such as UbcD1, and/or the caspase Dronc. Of the known proteins that are preferentially trafficked to dendrites, these molecules have not been implicated but warrant further investigation. Second, it is also possible that activated Dronc, or another p35-resistant protease activated by Dronc, could cleave a dendrite-specific substrate. Examples are now emerging from other cellular systems, such as in sperm formation and border cell migration, in which caspases can participate in cleavage of proteins not resulting in apoptosis. Third, the dendritic pruning program takes place during drastic environmental changes that include concurrent degradation and regrowth of the overlying epidermis, activation of extracellular matrix metalloproteases, and blood phagocytes. These environmental cues likely complement the neuronal intrinsic pruning programs, but their exact relationships are not known. Experiments addressing these and other possible mechanisms should provide a greater insight into how the large-scale remodeling of C4da neuron dendrites is achieved (Kuo, 2006).

In vertebrates, the UPS pathway has been implicated in Wallerian degeneration of severed axons. In the fly, mushroom body γ neurons undergo extensive remodeling of their processes during metamorphosis. The initial stages of axon pruning in these mushroom body neurons closely resemble Wallerian degeneration, and the UPS again plays a critical role. To date, the specific ubiquitin-conjugating enzymes and ligases that mediate target protein degradation have not been identified in these systems. It will be interesting to see whether the UbcD1-DIAP1-Dronc pathway implicated in C4da neuron dendrite pruning also participates in remodeling/degradation of neuronal processes in other systems. It seems likely that more than one pathway would be employed in remodeling different neurons; a previous study excluded UbcD1 as a possible ubiquitin-conjugating enzyme regulating mushroom body γ neuron remodeling, and normal remodeling of mushroom body neuron processes in is seen dronc mutant MARCM clones during metamorphosis (Kuo, 2006).

A multilayered regulatory machinery for remodeling neurons, as uncovered in this study for C4da neurons, offers versatility and flexibility. It is conceivable that another ubiquitin ligase/caspase pair may function in an analogous UPS pathway during mushroom body neuron remodeling, potentially affording differential regulation of neuronal remodeling. Although pharmacological inhibition of mammalian caspases showed no effect on Wallerian degeneration, it would be important to assess the in vivo effectiveness of the inhibitors against a comprehensive panel of caspases. Moreover, a dual control mechanism, similar to what is proposed for C4da neuron remodeling, may coordinately regulate UPS and another protease that executes axon degradation. Conceivably, instead of having the target of the UPS directly involved in maintaining dendrite/axon stability, the executor of neuronal process degradation may involve a different protease: in the case of C4da neurons it is the caspase Dronc, and in Wallerian degeneration the relevant protease might be the Ca2+-responsive calpain. Future experiments along these lines of thinking may accelerate the identification of specific ubiquitinating enzymes involved in other areas of developmental neuronal remodeling and in diseases where the UPS pathway has been implicated. As target-specific E3 ligases are excellent candidates for pharmaceutical intervention, this approach may also help to find effective treatments for developmental and neurodegenerative diseases that involve degeneration of neuronal processes (Kuo, 2006).

Thin is required for cell death in the Drosophila abdominal muscles by targeting DIAP1

In holometabolous insects, developmentally controlled programmed cell death (PCD) is a conserved process that destroys a subset of larval tissues for the eventual creation of new adult structures. This process of histolysis is relatively well studied in salivary gland and midgut tissues, while knowledge concerning larval muscle destruction is limited. This study examined the histolysis of a group of Drosophila larval abdominal muscles called the dorsal external oblique muscles (DEOMs). Previous studies have defined apoptosis as the primary mediator of DEOM breakdown, whose timing is controlled by ecdysone signaling. However, very little is known about other factors that contribute to DEOM destruction. In this paper, the role of thin (tn), which encodes for the Drosophila homolog of mammalian TRIM32, was examined in the regulation of DEOM histolysis. Loss of Tn blocks DEOM degradation independent of ecdysone signaling. Instead, tn genetically functions in a pathway with the death-associated inhibitor of apoptosis (DIAP1), Dronc, and death-associated APAF1-related killer (Dark) to regulate apoptosis. Importantly, blocking Tn results in the absence of active Caspase-3 immunostaining, upregulation of DIAP1 protein levels, and inhibition of Dronc activation. DIAP1 and Dronc mRNA levels are not altered in tn mutants, showing that Tn acts post-transcriptionally on DIAP1 to regulate apoptosis. This study also found that the RING domain of Tn is required for DEOM histolysis as loss of this domain results in higher DIAP1 levels. Together, these results suggest that the direct control of DIAP1 levels, likely through the E3 ubiquitin ligase activity of Tn, provides a mechanism to regulate caspase activity and to facilitate muscle cell death (Vishal, 2018).

PCD is required for the destruction of certain larval tissues during metamorphosis. A previous study established that histolysis of the abdominal muscles is regulated by apoptosis, while blocking autophagy does not affect muscle breakdown. In addition to the ecdysone receptor, only a handful of nuclear proteins are known to function in dorsal exterior oblique muscle (DEOM) histolysis. Loss of East results in a partial block in DEOM degeneration, whereas premature muscle destruction is observed in muscles that lack Chromator. Moreover, the two nuclear receptors, FTZ-F1 and HR39, antagonistically function to regulate the timing of DEOM histolysis. This study has further identified Tn as a novel protein in pupal muscle remodeling. However, loss of Tn does not affect salivary gland and midgut histolysis, highlighting an exclusive muscle role for Tn during Drosophila metamorphosis (Vishal, 2018).

Genetic assays demonstrate that tn functions with core components of the cell death machinery to regulate DEOM destruction. It was surprising that inhibition of apoptotic activity in Dark or Dronc mutants was not sufficient to completely block histolysis by 24 h APF. One explanation is the existence of additional cell death mechanisms other than apoptosis. While histolysing DEOMs contained autophagic vesicles, a reduction in autophagy components did not block or delay muscle degradation at 8 h APF. Tests were performed to see if a decrease in Tn-mediated apoptosis could sensitize muscle cells to initiate autophagy as a compensatory mechanism to assure cell death. However, this does not seem to be the case as RNAi knockdown of Atg1, Atg5, or Atg18 does not further block DEOM histolysis in a tn RNAi background. A second explanation is that the hypomorphic nature of these alleles may not completely abrogate Dark and Dronc function. Alternatively, additional effector caspases, including Dcp-1, Decay, and/or Damm, may be operating in the latter stages of DEOM histolysis since these caspases may function redundantly or act independent of the DIAP1-Drice axis (Vishal, 2018).

More than a partial block in DEOM histolysis was expected upon manipulation of DIAP1 (i.e., DIAP1 OE alone or tn RNAi+DIAP1 OE) at 12 h APF. It is possible that normal or overexpressed DIAP1 levels in the DEOMs are not high enough to block apoptosis, especially using RNAi approaches to reduce Tn levels. However, the use of tn-null alleles clearly shows a complete block in muscle degradation and a corresponding inhibition of active Dronc. Seemingly a delicate balance exists to regulate mRNA and protein expression, as well as protein turnover and proteolytic processing of active caspases. Cells must normally prevent cell death and only activate the apoptotic cascade upon a commitment to die. Thus, threshold levels of caspase activity must be reached for this terminal fate. There is evidence for stage or tissue-specific differential sensitivity to pro-apoptotic factors. Early L3 individuals are resistant to apoptosis, while wandering L3 larvae have elevated levels of Dark, Dronc, and Drice that are sufficient to trigger cell death under the appropriate stimuli. A model is proposed whereby Tn, through its RING domain, normally ubiquitinates DIAP1 for delivery to the proteasome during DEOM histolysis. This degradation of DIAP1 relieves Dronc inhibition, thereby initiating the caspase cascade for the execution of cell death. A general reduction in Tn, or loss of RING domain activity, prevents the addition of ubiquitin moieties and causes an increase in DIAP1 levels, effectively blocking cell death by limiting caspase activity (Vishal, 2018).

Numerous roles have been identified for mammalian TRIM32 in normal and cancerous cells. In muscle, mutations in the NHL repeats result in limb-girdle muscular dystrophy type 2H or sarcotubular myopathy. Several structural muscle proteins are targets of TRIM32 activity, including tropomyosin, desmin, α-actinin, and dysbindin. However, it is not yet clear if regulation of these muscle substrates contributes to normal muscle physiology, is required to prevent atrophy, or plays a critical role in disease pathology. The TRIM32-mediated degradation of additional protein substrates, including p53, Abi2, Piasy, and the X-linked IAP (XIAP), contribute to oncogenic or tumor suppressor activities that either confer resistance or susceptibility to apoptosis. Tumor necrosis factor-α can trigger death receptor-mediated apoptosis through the regulation of XIAP activity. TRIM32 colocalizes and directly interacts with XIAP in human kidney epithelial cells (HEK293). Moreover, TRIM32 induces apoptosis through the direct ubiquitination and subsequent protein turnover of XIAP degradation. This control of apoptotic cell death mirrors the current genetic results, strongly suggesting that this TRIM32-mediated regulation of IAP family members may be a conserved mechanism to regulate apoptosis. It would be interesting to further investigate if Tn and mammalian TRIM32 regulates apoptotic decisions in other contexts of muscle development and/or disease (Vishal, 2018).

An ADAMTS Sol narae is required for cell survival in Drosophila

Cell survival is essential for all living organisms to cope against multiple environmental insults. Intercellular signaling between dying and surviving cells plays an important role to ensure compensatory proliferation, preventing tissue loss after environmental stresses. This study shows that Sol narae (Sona), a Disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) in Drosophila is required for cell survival. sona exhibited a positive genetic interaction with Death-associated inhibitor of apoptosis 1 (Diap1), and a negative genetic interaction with reaper (rpr). Transcription patterns of sona, Diap1, and rpr genes in the pouch region of wing discs were coordinately changed after irradiation. Interestingly, there was a negative correlation in the expression levels of Sona and DIAP1, and both cell types, one with high Sona level and the other with high Diap1 level, were resistant to irradiation-induced cell death. The sona-expressing cells rarely entered into cell cycle themselves but promoted the nearby cells to proliferate in irradiation conditions. These sona-expressing cells are able to upregulate Cyclin D (Cyc D) and increase tissue size. Furthermore, transient Sona overexpression increased survival rate and promoted development of flies in irradiation conditions. It is proposed that the two types of radiation-resistant cells, one with high Sona level and the other with high Diap1 level, communicate with dying cells and between each other for cell survival and proliferation in response to irradiation (Tsogtbaatar, 2019).

This study has shown that Sona functions in two different ways. First, Sona is required cell autonomously for cell survival, and the level of sona transcription correlates with the degree of cell survival under the irradiated condition. Cells expressing a high level of either sona or Diap1 are resistant to cell death, so there are at least two different types of surviving cells in wing discs. Second, Sona induces cell proliferation cell non-autonomously. Transient expression of sona upregulated Cyc D in neighboring cells but not in sona-expressing cells, suggesting that radiation-resistant sona-expressing cells secrete signaling molecule(s) that upregulates Cyc D in neighboring cells. In conjunction with genetic interactions between sona, Diap1 and rpr, cells expressing sona may dynamically interplay with other cells that express Diap1 and rpr for damage control (Tsogtbaatar, 2019).

Sona is expressed in the patchy pattern, and sona clones with different sizes were generated in the wing pouch region. It is speculated that the large sona clones are generated where sona is not expressed. Similar result was reported in the loss-of-function clone analysis of the vestigial (vg) gene that is essential for survival and proliferation of wing disc cells. vg RNAi clones are generated where Vg is not expressed or expressed at a low level in wing discs. That is, the loss-of-function vg clones are rarely formed where Vg is required for cell survival and proliferation. Indeed, Vg drives cell cycle progression by inducing dE2F1 gene whose product is essential for G1 to S transition in cell cycle. Formation of clones with different sizes may be a general phenomenon for the genes that are expressed unevenly and are required for cell proliferation or cell survival. Varied levels of sona and vg expression in different parts of wing discs may be required to create regional differences in growth rate in order to form wings with proper size and shape (Tsogtbaatar, 2019).

What are the distinct functions of sona-expressing cells? In normal conditions, sona is expressed in a widely varing level in a patchy pattern, and acts as a signal to promote cell proliferation for wing development. In irradiated conditions, the transcriptional level of sona dramatically increased among sona-expressing cells that may be required to cope against extensive cell death. This irradiation generated two major cell groups in the wing pouch. The first group of cells are determined to grow and proliferate or, alternatively, to die upon a high level of irradiation. The second group of cells are resistant to irradiation and send out signals to regulate the first group of cells. The first group of cells should express proteins for cell growth, cell proliferation, cell survival and cell death including Diap1 and Rpr. The second group of cells should have capacity to sense the changes in environment and send out signals to regulate the first group of cells. In this study, Sona was identified as one of the proteins expressed in the second group of cells in a patchy pattern. Interestingly, a phosphorylated form of ribosomal protein S6 (pS6) that should be regulated in the first group of cells is also present in a patchy pattern. It is speculated that pS6 and sona may express in the first and the second groups of cells, respectively (Tsogtbaatar, 2019).

This study showed that Sona induces Cyc D in nearby cells, which is consistent with the finding that overexpressed Sona increases tissue size. Cyc D is known as a target protein of Wnt signaling in mammals, and acts in a G1 phase of cell cycle that responds to mitogens. In flies, overexpression of Cyc D/Cdk4 accelerates cell division in proliferating wing disc cells while increasing the cell size in post-mitotic cells by endoreplication. Because Cyc D is required for cell division of surviving cells, it should be upregulated in Diap1-expressing cells. Indeed, Diap1 is induced by Yorkie and STAT in Hippo pathway and JAK-STAT pathway, respectively, and these two pathways are essential for cell proliferation in a cell autonomous manner. An important question is the identity of the direct signal(s) that upregulate Cyc D. Secreted Sona may act as a direct signal or, as a metalloprotease, may modulate the activity of other signaling molecules such as Wg. It has been reported that irradiation activates a damage-activated enhancer BRV118 of the wg gene. Therefore, irradiation changes the transcription pattern of both sona and wg, and may coordinate the functions of Sona and Wg for induction of Cyc D, although Sona may activate yet another signaling molecules (Tsogtbaatar, 2019).

An interesting new mechanism to repair tissue damage has been recently discovered, in which cells of the presumptive hinge region in the wing disc migrate into the pouch region after irradiation. These hinge cells also migrate into the pouch region when the pouch cells are killed by hid expression. These hinge cells are resistant to cell death due to the activation of Wg signaling and STAT signaling that suppress the transcription of rpr52. Resistance to irradiation and lack of rpr expression are also characteristics of sona-expressing cells, suggesting that the sona-expressing cells in the wing pouch may be functionally related to the radiation-resistant hinge cells. Because intense irradiation physically induces DNA breakage even in these radiation-resistant cells, the mechanism by which these sona-expressing cells cope against DNA damage is an important question to be explored (Tsogtbaatar, 2019).

Several mammalian metalloproteases in ECM are also shown to prevent apoptosis. Overexpression of MMP-15 (matrix metalloprotease-15) prevents apoptosis of Hela and human adenocarcinoma. Furthermore, ADAM-12 increases apoptosis of stromal cell but decreases that of tumor cells. In ADAMTS family, ADAMTS is shown to be required for cell survival. ADAMTS is mutated in belted (bt) mice that show cell death of melanoblast. Sona is also involved in cell survival by increasing resistance against irradiation and by promoting cell proliferation of neighboring cells. Identification of Sona substrate and its function will greatly help understand the role of Sona in cell survival, which is currently ongoing in our laboratory (Tsogtbaatar, 2019). .

Control of hormone-driven organ disassembly by ECM remodeling and Yorkie-dependent apoptosis

Epithelia grow and shape into functional structures during organogenesis. Although most of the focus on organogenesis has been drawn to the building of biological structures, the disassembly of pre-existing structures is also an important event to reach a functional adult organ. Examples of disassembly processes include the regression of the Mullerian or Wolffian ducts during gonad development and mammary gland involution during the post-lactational period in adult females. To date, it is unclear how organ disassembly is controlled at the cellular level. This study follows the Drosophila larval trachea through metamorphosis and shows that its disassembly is a hormone-driven and precisely orchestrated process. It occurs in two phases: first, remodeling of the apical extracellular matrix (aECM), mediated by matrix metalloproteases and independent of the actomyosin cytoskeleton, results in a progressive shortening of the entire trachea and a nuclear-to-cytoplasmic relocalization of the Hippo effector Yorkie (Yki). Second, a decreased transcription of the Yki target, Diap1, in the posterior metameres and the activation of caspases result in the apoptotic loss of the posterior half of the trachea while the anterior half escapes cell death. Thus, this work unravels a mechanism by which hormone-driven ECM remodeling controls sequential tissue shortening and apoptotic cell removal through the transcriptional activity of Yki, leading to organ disassembly during animal development (Fraire-Zamora, 2021).

This study report how a functional organ, the larval trachea of Drosophila melanogaster, undergoes a hormone-driven disassembly during metamorphosis. The dorsal trunks of the trachea shorten modularly in two sequential phases: (1) an initial progressive phase of a controlled reduction in the trachea length, involving aECM remodeling and cellular shape changes and, as a consequence, (2) Yki inactivation results in a decreased transcription of its target gene (and apoptosis inhibitor) Diap1 in the posterior metameres, resulting in their loss through cell death (Fraire-Zamora, 2021).

It was found that the activation of apoptosis results in the disassembly and loss of only the posterior metameres (Tr6-Tr8). Why are anterior metameres (Tr3-Tr5) not affected? This is an intriguing observation because most of the signaling inputs occur along the whole dorsal trunk. These results suggest that the anterior metameres are 'protected' or 'desensitized' against the signals that occur along the dorsal trunks. The results on downregulation of AbdB suggest that Hox genes play a role in the differential response between anterior and posterior metameres during dorsal trunk disassembly. However, it cannot be excluded that other elements might also contribute, such as the proximity of pools of adult progenitor cells to the anterior metameres. Whether the anterior metameres are protected against MMP-1 activity and cell death through a compartmentalization of the trachea via Hox genes or through signals from the progenitor cells is a matter of future work (Fraire-Zamora, 2021).

The Hpo signaling pathway and its effector Yki/YAP are conserved both in invertebrates and vertebrates where they regulate organ size through the transcriptional control of genes related to proliferation and cell death. While work in mammalian cell cultures has shown that ECM-related inactivation of YAP can lead to an increase in apoptosis, most of the focus has concentrated on how the Hpo signaling pathway controls proliferation, with very few examples on the activation of apoptosis (Fraire-Zamora, 2021).

The current results unveil a new role of the Hpo pathway in the hormone-driven disassembly of an organ through an ECM-triggered inactivation of the Hpo effector Yki, resulting in the triggering of apoptosis on the posterior end of the trachea. Given the conservation of the Hpo pathway, it is speculated that its role in organ disassembly could be of general use in some of the widespread events leading to organ involution (i.e., the controlled regression or shrinkage of an organ) during embryonic development or in homeostatic processes during adult life or aging (Fraire-Zamora, 2021).


cDNA clone length - 2015

Bases in 5' UTR - 430

Exons - 2

Bases in 3' UTR - 267


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).

Death-associated inhibitor of apoptosis 1: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 5 December 2023

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