Drosophila as a model for unfolded protein response research

Abstract

Endoplasmic Reticulum (ER) is an organelle where most secretory and membrane proteins are synthesized, folded, and undergo further maturation. As numerous conditions can perturb such ER function, eukaryotic cells are equipped with responsive signaling pathways, widely referred to as the Unfolded Protein Response (UPR). Chronic conditions of ER stress that cannot be fully resolved by UPR, or conditions that impair UPR signaling itself, are associated with many metabolic and degenerative diseases. In recent years, Drosophila has been actively employed to study such connections between UPR and disease. Notably, the UPR pathways are largely conserved between Drosophila and humans, and the mediating genes are essential for development in both organisms, indicating their requirement to resolve inherent stress. By now, many Drosophila mutations are known to impose stress in the ER, and a number of these appear similar to those that underlie human diseases. In addition, studies have employed the strategy of overexpressing human mutations in Drosophila tissues to perform genetic modifier screens. The fact that the basic UPR pathways are conserved, together with the availability of many human disease models in this organism, makes Drosophila a powerful tool for studying human disease mechanisms.

Fig. 1.. Regulation and detection of the IRE1/XBP1 pathway. (A) A schematic diagram of the IRE1/XBP1 pathway in Drosophila. IRE1 is an ER stress sensor that directly binds to misfolded peptides in the ER lumen. Upon detecting ER stress, IRE1 activates its RNase domain on the cytoplasmic side. IRE1 works together with a tRNA ligase to catalyze the splicing of XBP1 mRNA. The product of this spliced isoform acts as a transcription factor that induces ER quality control genes. In addition, IRE1 promotes the decay of many mRNAs associated with the ER. (B) The XBP1-GFP reporter used to detect IRE1 activity in vivo. As XBP1 mRNA splicing by IRE1 shifts the reading frame, an XBP1-GFP fusion transgene was designed to have GFP expressed in frame only when IRE1-mediated splicing occurs.

Fig. 2.. Regulation of the PERK/ATF4 pathway in Drosophila. (A) A schematic diagram of the pathway initiated by the eIF2alpha kinases, PERK and GCN2. These kinases are activated by distinct types of stress and phosphorylate eIF2alpha. This results in the overall translation attenuation, but at least two transcripts in Drosophila enhance their translation during such conditions: ATF4 is a transcription factor that induces stress response genes, and PPP1R15 is a phosphatase subunit that helps to de-phosphorylate eIF2alpha as a feedback mechanism. (B) uORFs in the 5’ UTR allow enhanced ATF4 synthesis under conditions of eIF2alpha phosphorylation. ATF4 5’ UTR has multiple uORFs, and only two are shown for simplicity. eIF2alpha helps to charge 40S ribosomes with initiator methionyl tRNA after the synthesis of uORF1. When eIF2alpha is active, 40S ribosomes efficiently recognize uORF2 for translation. uORF2 overlaps with ATF4 ORF, and interferes with ATF4 ORF translation. When eIF2alpha is phosphorylated, 40S ribosome’s ability to recognize the uORF2 is compromised, bypassing its AUG to allow the recognition of the ATF4 ORF.