Impaired proteasomal degradation enhances autophagy via hypoxia signaling in Drosophila

Abstract

BackgroundTwo pathways are responsible for the majority of regulated protein catabolism in eukaryotic cells: the ubiquitin-proteasome system (UPS) and lysosomal self-degradation through autophagy. Both processes are necessary for cellular homeostasis by ensuring continuous turnover and quality control of most intracellular proteins. Recent studies established that both UPS and autophagy are capable of selectively eliminating ubiquitinated proteins and that autophagy may partially compensate for the lack of proteasomal degradation, but the molecular links between these pathways are poorly characterized.ResultsHere we show that autophagy is enhanced by the silencing of genes encoding various proteasome subunits (α, β or regulatory) in larval fat body cells. Proteasome inactivation induces canonical autophagy, as it depends on core autophagy genes Atg1, Vps34, Atg9, Atg4 and Atg12. Large-scale accumulation of aggregates containing p62 and ubiquitinated proteins is observed in proteasome RNAi cells. Importantly, overexpressed Atg8a reporters are captured into the cytoplasmic aggregates, but these do not represent autophagosomes. Loss of p62 does not block autophagy upregulation upon proteasome impairment, suggesting that compensatory autophagy is not simply due to the buildup of excess cargo. One of the best characterized substrates of UPS is the α subunit of hypoxia-inducible transcription factor 1 (HIF-1α), which is continuously degraded by the proteasome during normoxic conditions. Hypoxia is a known trigger of autophagy in mammalian cells, and we show that genetic activation of hypoxia signaling also induces autophagy in Drosophila. Moreover, we find that proteasome inactivation-induced autophagy requires sima, the Drosophila ortholog of HIF-1α.ConclusionsWe have characterized proteasome inactivation- and hypoxia signaling-induced autophagy in the commonly used larval Drosophila fat body model. Activation of both autophagy and hypoxia signaling was implicated in various cancers, and mutations affecting genes encoding UPS enzymes have recently been suggested to cause renal cancer. Our studies identify a novel genetic link that may play an important role in that context, as HIF-1α/sima may contribute to upregulation of autophagy by impaired proteasomal activity.

A visual screen for diet-regulated proteins in the Drosophila ovary using GFP protein trap lines

“…The reduced expression of me31B:GFP in germline cysts under starvation might accompany reduced rate of transport into the oocyte. CG5059 encodes the ortholog of mammalian BNIP3 (Low et al, 2013), which belongs to the Bcl-2 family and is involved in necrosis, apoptosis and autophagy (Burton and Gibson, 2009; Low et al, 2013). CG5059::GFP shows increased expression in subcellular structures within cystoblasts and germline cysts of females on a poor relative to rich diet (Fig.…”

Hypoxia Potentiates LPS-Mediated Cytotoxicity of BV2 Microglial Cells In Vitro by Synergistic Effects on Glial Cytokine and Nitric Oxide System

“…35 Thereby, the chronology of microglial activation and hypoxia is crucial as hypoxic preconditioning has been shown be neuroprotective in rodent models of perinatal cerebral hypoxic ischemia 36 and in cell culture experiments. 37 Irrespectively, hypoxia-enhanced microglial cytotoxicity seems to be induced by activation of apoptotic processes, 38 inhibition of proteasomal degradation of cytokine precursors, 22 and stimulation of further cytotoxic mediators that strengthens TNF-α cytotoxicity up to 200 folds. 39 In conclusion, our data show, that recurrent LPS-induced expression of IL-1β, IL-6, and TNF-α extending LPS activation may establish a positive autocrine feedback loop, which provokes long-lasting NO secretion.…”

Assays to monitor autophagy in Drosophila

“…However, the use of lysosomal inhibitors is poorly compatible with studies involving intact larvae or adult flies. Although prolonged feeding of chloroquine (24 h) has been shown to inhibit lysosomal acidity in the larval fat body [35, 36], incubations of this time scale can cause secondary effects resulting from the accumulation of undigested lysosomal cargo [5]. Therefore additional assays of autophagic flux have been developed or adapted for use in Drosophila.…”

“…Most cells undergoing autophagy will display a combination of both green+red autophagosomes and red-only autolysosomes, the latter of which tend to predominate at later timepoints. Juhasz and colleagues have used chloroquine feeding to show that the loss of GFP fluorescence duly reflects the low pH of the lysosome [35, 36]. This marker thus provides evidence of both biogenesis of autophagosomes and their successful fusion with a properly acidified lysosomal compartment.…”