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Bip-Yorkie interaction determines oncogenic and tumor-suppressive roles of Ire1/Xbp1s activation

Yang, Shuai; Jiang, Hua; Bian, Weixiang; Xu, Wenyan; Guo, Yifan; Song, Sha; Zheng, Jiadong; Kuang, Xiaoyu; Wu, Chenxi; Ding, Xiang; Guo, Xiaowei; Xue, Lei; Yu, Zijing; Zhang, Yongdeng; Ryoo, Hyung Don; Li, Xu; Ma, Xianjue
Unfolded protein response (UPR) is the mechanism by which cells control endoplasmic reticulum (ER) protein homeostasis. ER proteostasis is essential to adapt to cell proliferation and regeneration in development and tumorigenesis, but mechanisms linking UPR, growth control, and cancer progression remain unclear. Here, we report that the Ire1/Xbp1s pathway has surprisingly oncogenic and tumor-suppressive roles in a context-dependent manner. Activation of Ire1/Xbp1s up-regulates their downstream target Bip, which sequesters Yorkie (Yki), a Hippo pathway transducer, in the cytoplasm to restrict Yki transcriptional output. This regulation provides an endogenous defensive mechanism in organ size control, intestinal homeostasis, and regeneration. Unexpectedly, Xbp1 ablation promotes tumor overgrowth but suppresses invasiveness in a Drosophila cancer model. Mechanistically, hyperactivated Ire1/Xbp1s signaling in turn induces JNK-dependent developmental and oncogenic cell migration and epithelial-mesenchymal transition (EMT) via repression of Yki. In humans, a negative correlation between XBP1 and YAP (Yki ortholog) target gene expression specifically exists in triple-negative breast cancers (TNBCs), and those with high XBP1 or HSPA5 (Bip ortholog) expression have better clinical outcomes. In human TNBC cell lines and xenograft models, ectopic XBP1s or HSPA5 expression alleviates tumor growth but aggravates cell migration and invasion. These findings uncover a conserved crosstalk between the Ire1/Xbp1s and Hippo signaling pathways under physiological settings, as well as a crucial role of Bip-Yki interaction in tumorigenesis that is shared from Drosophila to humans.
PMCID:9586321
PMID: 36215479
ISSN: 1091-6490
CID: 5351922

A Protein-trap allele reveals roles for Drosophila ATF4 in photoreceptor degeneration, oogenesis and wing development

Vasudevan, Deepika; Katow, Hidetaka; Huang, Huai-Wei; Tang, Grace; Ryoo, Hyung Don
Metazoans have evolved various quality control mechanisms to cope with cellular stress inflicted by external and physiological conditions. ATF4 is a major effector of the Integrated Stress Response (ISR), an evolutionarily conserved pathway that mediates adaptation to various cellular stressors. Loss of function of Drosophila ATF4, encoded by the gene cryptocephal (crc), results in lethality during pupal development. The roles of crc in Drosophila disease models and in adult tissue homeostasis thus remain poorly understood. Here, we report that a protein-trap MiMIC insertion in the crc locus generates a crc-GFP fusion protein that allows visualization of crc activity in vivo. This allele also acts as a hypomorphic mutant that uncovers previously unknown roles for crc. Specifically, the crc protein-trap line shows crc-GFP induction in a Drosophila model for Retinitis Pigmentosa (RP). This crc allele renders flies more vulnerable to amino acid deprivation and age-dependent retinal degeneration. These mutants also show defects in wing veins and oocyte maturation. Together, our data reveal previously unknown roles for crc in development, cellular homeostasis and photoreceptor survival.
PMID: 34919148
ISSN: 1754-8411
CID: 5109872

Drosophila Unfolded Protein Response (UPR) Assays In Vitro and In Vivo

Katow, Hidetaka; Vasudevan, Deepika; Ryoo, Hyung Don
Wildtype or mutant proteins expressed beyond the capacity of a cell's protein folding system could be detrimental to general cellular function and survival. In response to misfolded protein overload in the endoplasmic reticulum (ER), eukaryotic cells activate the Unfolded Protein Response (UPR) that helps cells restore protein homeostasis in the endoplasmic reticulum (ER). As part of the UPR, cells attenuate general mRNA translation and activate transcription factors that induce stress-responsive gene expression.UPR signaling draws research interest in part because conditions that cause chronic protein misfolding in the ER or those that impair UPR signaling underlie several diseases including neurodegeneration, diabetes, and cancers. Model organisms are frequently employed in the field as the UPR pathways are generally well-conserved throughout phyla. Here, we introduce experimental procedures to detect UPR in Drosophila melanogaster.
PMID: 34985706
ISSN: 1940-6029
CID: 5107162

Drosophila fabp is required for light-dependent Rhodopsin-1 clearance and photoreceptor survival

Huang, Huai-Wei; Ryoo, Hyung Don
Rhodopsins are light-detecting proteins coupled with retinal chromophores essential for visual function. Coincidentally, dysfunctional rhodopsin homeostasis underlies retinal degeneration in humans and model organisms. Drosophila ninaEG69D mutant is one such example, where the encoded Rh1 protein imposes endoplasmic reticulum (ER) stress and causes light-dependent retinal degeneration. The underlying reason for such light-dependency remains unknown. Here, we report that Drosophila fatty acid binding protein (fabp) is a gene induced in ninaEG69D/+ photoreceptors, and regulates light-dependent Rhodopsin-1 (Rh1) protein clearance and photoreceptor survival. Specifically, our photoreceptor-specific gene expression profiling study in ninaEG69D/+ flies revealed increased expression of fabp together with other genes that control light-dependent Rh1 protein degradation. fabp induction in ninaEG69D photoreceptors required vitamin A and its transporter genes. In flies reared under light, loss of fabp caused an accumulation of Rh1 proteins in cytoplasmic vesicles. The increase in Rh1 levels under these conditions was dependent on Arrestin2 that mediates feedback inhibition of light-activated Rh1. fabp mutants exhibited light-dependent retinal degeneration, a phenotype also found in other mutants that block light-induced Rh1 degradation. These observations reveal a previously unrecognized link between light-dependent Rh1 proteostasis and the ER-stress imposing ninaEG69D mutant that cause retinal degeneration.
PMID: 34714826
ISSN: 1553-7404
CID: 5042882

The transcription factor Xrp1 is required for PERK-mediated antioxidant gene induction in Drosophila

Brown, Brian; Mitra, Sahana; Roach, Finnegan D; Vasudevan, Deepika; Ryoo, Hyung Don
PERK is an endoplasmic reticulum (ER) transmembrane sensor that phosphorylates eIF2α to initiate the Unfolded Protein Response (UPR). eIF2α phosphorylation promotes stress-responsive gene expression most notably through the transcription factor ATF4 that contains a regulatory 5' leader. Possible PERK effectors other than ATF4 remain poorly understood. Here, we report that the bZIP transcription factor Xrp1 is required for ATF4-independent PERK signaling. Cell-type-specific gene expression profiling in Drosophila indicated that delta-family glutathione-S-transferases (gstD) are prominently induced by the UPR-activating transgene Rh1G69D. Perk was necessary and sufficient for such gstD induction, but ATF4 was not required. Instead, Perk and other regulators of eIF2α phosphorylation regulated Xrp1 protein levels to induce gstDs. The Xrp1 5' leader has a conserved upstream Open Reading Frame (uORF) analogous to those that regulate ATF4 translation. The gstD-GFP reporter induction required putative Xrp1 binding sites. These results indicate that antioxidant genes are highly induced by a previously unrecognized UPR signaling axis consisting of PERK and Xrp1.
PMCID:8514241
PMID: 34605405
ISSN: 2050-084x
CID: 5039492

The role of Ire1 in Drosophila eye pigmentation revealed by an RNase dead allele

Mitra, Sahana; Ryoo, Hyung Don
Ire1 is an endoplasmic reticulum (ER) transmembrane RNase that cleaves substrate mRNAs to help cells adapt to ER stress. Because there are cell types with physiological ER stress, loss of Ire1 results in metabolic and developmental defects in diverse organisms. In Drosophila, Ire1 mutants show developmental defects at early larval stages and in pupal eye photoreceptor differentiation. These Drosophila studies relied on a single Ire1 loss of function allele with a Piggybac insertion in the coding sequence. Here, we report that an Ire1 allele with a specific impairment in the RNase domain, H890A, unmasks previously unrecognized Ire1 phenotypes in Drosophila eye pigmentation. Specifically, we found that the adult eye pigmentation is altered, and the pigment granules are compromised in Ire1H890A homozygous mosaic eyes. Furthermore, the Ire1H890A mutant eyes had dramatically reduced Rhodopsin-1 protein levels. Drosophila eye pigment granules are most notably associated with late endosome/lysosomal defects. Our results indicate that the loss of Ire1, which would impair ER homeostasis, also results in altered adult eye pigmentation.
PMID: 34265355
ISSN: 1095-564x
CID: 4938882

Periphery signals generated by Piezo-mediated stomach stretch and Neuromedin-mediated glucose load regulate the Drosophila brain nutrient sensor

Oh, Yangkyun; Lai, Jason Sih-Yu; Min, Soohong; Huang, Huai-Wei; Liberles, Stephen D; Ryoo, Hyung Don; Suh, Greg S B
Nutrient sensors allow animals to identify foods rich in specific nutrients. The Drosophila nutrient sensor, diuretic hormone 44 (DH44) neurons, helps the fly to detect nutritive sugar. This sensor becomes operational during starvation; however, the mechanisms by which DH44 neurons or other nutrient sensors are regulated remain unclear. Here, we identified two satiety signals that inhibit DH44 neurons: (1) Piezo-mediated stomach/crop stretch after food ingestion and (2) Neuromedin/Hugin neurosecretory neurons in the ventral nerve cord (VNC) activated by an increase in the internal glucose level. A subset of Piezo+ neurons that express DH44 neuropeptide project to the crop. We found that DH44 neuronal activity and food intake were stimulated following a knockdown of piezo in DH44 neurons or silencing of Hugin neurons in the VNC, even in fed flies. Together, we propose that these two qualitatively distinct peripheral signals work in concert to regulate the DH44 nutrient sensor during the fed state.
PMID: 34015253
ISSN: 1097-4199
CID: 4877522

Translational induction of ATF4 during integrated stress response requires noncanonical initiation factors eIF2D and DENR

Vasudevan, Deepika; Neuman, Sarah D; Yang, Amy; Lough, Lea; Brown, Brian; Bashirullah, Arash; Cardozo, Timothy; Ryoo, Hyung Don
The Integrated Stress Response (ISR) helps metazoan cells adapt to cellular stress by limiting the availability of initiator methionyl-tRNA for translation. Such limiting conditions paradoxically stimulate the translation of ATF4 mRNA through a regulatory 5' leader sequence with multiple upstream Open Reading Frames (uORFs), thereby activating stress-responsive gene expression. Here, we report the identification of two critical regulators of such ATF4 induction, the noncanonical initiation factors eIF2D and DENR. Loss of eIF2D and DENR in Drosophila results in increased vulnerability to amino acid deprivation, susceptibility to retinal degeneration caused by endoplasmic reticulum (ER) stress, and developmental defects similar to ATF4 mutants. eIF2D requires its RNA-binding motif for regulation of 5' leader-mediated ATF4 translation. Consistently, eIF2D and DENR deficient human cells show impaired ATF4 protein induction in response to ER stress. Altogether, our findings indicate that eIF2D and DENR are critical mediators of ATF4 translational induction and stress responses in vivo.
PMID: 32938929
ISSN: 2041-1723
CID: 4593222

Neuronally expressed anti-tau scFv prevents tauopathy-induced phenotypes in Drosophila models

Krishnaswamy, S; Huang, H-W; Marchal, I S; Ryoo, H D; Sigurdsson, E M
We have derived single-chain variable fragments (scFv) from tau antibody hybridomas and previously shown their promise as imaging diagnostic agents. Here, we examined the therapeutic potential of anti-tau scFv in transgenic Drosophila models that express in neurons wild-type (WT) human tau (htau) or the human tauopathy mutation R406W. scFv expressing flies were crossed with the tauopathy flies and analyzed. Overall, the survival curves differed significantly (p < .0001). Control flies not expressing htau survived the longest, whereas R406W expressing flies had the shortest live span, which was greatly prolonged by co-expressing the anti-tau scFv (p < .0001). Likewise, htau WT expressing flies had a moderately short live span, which was prolonged by co-expressing the anti-tau scFv (p < .01). In addition, the htau expression impaired wing expansion after eclosion (p < .0001), and caused progressive abdomen expansion (p < .0001). These features were more severe in htau R406W flies than in htau WT flies. Importantly, both phenotypes were prevented by co-expression of the anti-tau scFv (p < .01-0.0001). Lastly, brain analyses revealed scFv-mediated tau clearance (p < .05-0.01), and its prevention of tau-mediated neurotoxicity (p < .05-0.001). In summary, these findings support the therapeutic potential of an anti-tau scFv, including as gene therapies, and the use of Drosophila models for such screening.
PMID: 31982516
ISSN: 1095-953x
CID: 4293772

Corrigendum to "Triazolo[4,5-d]pyrimidines as validated general control nonderepressible 2 (GCN2) protein kinase inhibitors reduce growth of leukemia cells" [Comput. Struct. Biotechnol. J. 16 (2018) 350-360]

Lough, Lea; Sherman, Dan; Becerra-Flores, Manuel; Vasudevan, Deepika; Lavinda, Olga; Ni, Eric; Wang, Hong; Ryoo, Hyung Don; Tibes, Raoul; Cardozo, Timothy
[This corrects the article DOI: 10.1016/j.csbj.2018.09.003.].
PMID: 32435428
ISSN: 2001-0370
CID: 4444472