Identification of factors that function in Drosophila salivary gland cell death during development using proteomics

Abstract

Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation. Programmed cell death is critical to multi-cellular animal development and homeostasis, and misregulation of cell death can lead to disorders including autoimmunity and cancer.

Impaired proteasomal degradation enhances autophagy via hypoxia signaling in Drosophila

“…Indeed, genetic or pharmacological activation of autophagy facilitates aggregate clearance and neuronal function in disease models [1,5]. Chronic block of proteasomal degradation also leads to toxicity, ER stress and cell death in various mammalian and Drosophila cells, and proteasome inhibitors are in clinical use to trigger apoptosis of cancer cells in multiple myeloma patients [31-34]. While we found no evidence of cell death in larval Drosophila fat body upon silencing of genes encoding essential subunits of the proteasome or genetic activation of hypoxia signaling, these genetic manipulations strongly enhanced both basal and starvation-induced autophagy in these cells, and this effect was dependent on canonical autophagy genes Atg1, Vps34, Atg9, Atg4 and Atg12 .…”

Fibroblast autophagy in fibrotic disorders

“…Interestingly, the visualization of specific autophagic structures (autophagosomes) in dying cells has led to the belief that autophagy could be a non‐apoptotic form of programmed cell death . The best‐accepted example of autophagy as a cell death effector is the degradation of the salivary glands during pupal transition in Drosophila . In mammals, the possibility that autophagy could lead to cell death under persistent stress conditions appears still controversial and most evidence indicates that, at least in cells with intact apoptotic machinery, autophagy is primarily a pro‐survival rather than a pro‐death mechanism .…”

Mutants forDrosophilaIsocitrate Dehydrogenase 3b Are Defective in Mitochondrial Function and Larval Cell Death

“…This activation of rpr , dronc , and hid , as well as the partial repression of the apoptosis inhibitor diap1 by CBP ( Yin et al 2007 ), are key events in the initiation of salivary gland histolysis. To understand better the events of salivary gland cell death, large-scale genetic ( Wang et al 2008 ) and molecular ( Gorski et al 2003 ; Lee et al 2003 ; Martin et al 2007 ; McPhee et al 2013 ) screens have been conducted to identify additional genes that mediate the death response. The genes identified encode functionally diverse products, including an RNA helicase ( Ihry et al 2012 ), chromatin remodelers ( Ihry and Bashirullah 2014 ), an enzyme of the TCA cycle ( Wang et al 2010 ), subunits of the mediator complex ( Wang et al 2010 ), subunits of the cop9 signalsome ( McPhee et al 2013 ), several autophagy (Atg) proteins ( Berry and Baehrecke 2007 ), and several proteins of unknown function.…”