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Transcriptional stress responses induced by compartment-targeted misfolding-prone proteins in Drosophila

ABSTRACT: Loss of proteostasis is an aging hallmark and the culprit of many age-related diseases. Different cell compartments experience distinctive challenges to maintain protein quality control but it remains underexplored how subcellular proteostasis is regulated by aging. Here, by targeting the misfolding_x0002_prone FlucDM luciferase to the cytoplasm, mitochondria, and nucleus, we have established transgenic sensors to examine subcellular proteostasis in Drosophila. Analysis of detergent-insoluble and -soluble levels of compartment-targeted FlucDM variants indicates that thermal stress, cold shock, and endocrine signaling differentially affect subcellular proteostasis during aging. Moreover, aggregation-prone proteins that cause different neurodegenerative diseases induce a diverse range of outcomes on FlucDM insolubility, suggesting that subcellular proteostasis is deranged in a disease-specific manner. Further analyses with FlucDM and mass spectrometry indicate that pathogenic tauV337M regulates in opposite manners the solubility of protein sets located in distinct organelles. Altogether, compartment-targeted FlucDM variants pinpoint a diverse modulation of subcellular proteostasis by aging regulators.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE243224 | GEO | 2024/09/30

REPOSITORIES: GEO

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Project description:The unfolded protein response (UPR) maintains endoplasmic reticulum (ER) proteostasis through the activation of transcription factors such as XBP1s and ATF6. The functional consequences of these transcription factors for ER proteostasis remain poorly defined. Here, we describe methodology that enables orthogonal, small molecule-mediated activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the same cell independent of stress. We employ transcriptomics and quantitative proteomics to evaluate ER proteostasis network remodeling owing to the XBP1s and/or ATF6 transcriptional programs. Furthermore, we demonstrate that the three ER proteostasis environments accessible by activating XBP1s and/or ATF6 differentially influence the folding, trafficking, and degradation of destabilized ER client proteins without globally affecting the endogenous proteome. Our data reveal how the ER proteostasis network is remodeled by the XBP1s and/or ATF6 transcriptional programs at the molecular level and demonstrate the potential for selectively restoring aberrant ER proteostasis of pathologic, destabilized proteins through arm-selective UPR-activation. The unfolded protein response adapts endoplasmic reticulum (ER) proteostasis via stress-responsive transcription factors including XBP1s and ATF6. Here, R. Luke Wiseman and colleagues implement technology for the orthogonal, ligand-dependent activation of XBP1s and/or ATF6 in a single cell. They characterize how XBP1s and/or ATF6 activation impacts ER proteostasis pathway composition and function. Adapted ER environments influence the proteostasis of destabilized protein variants without affecting the endogenous proteome. The work informs the development of proteostasis environment-adapting therapeutics for protein misfolding-related diseases. In order to activate both XBP1s and ATF6 in the same cell, we incorporated DHFR.ATF6 and tet-inducible XBP1s into a HEK293T-REx cell line stably expressing the tet-repressor. The HEK293DYG control cell line expresses tet-inducible eGFP and DHFR.YFP and is used as a control to demonstrate that the addition of doxycycline (dox) and trimethoprim (TMP) do not induce UPR genes. HEK293DYG cells were treated for 12 h with vehicle or 1 μg/mL dox and 10 μM TMP in biological triplicate. Cells were harvested and RNA was extracted using the RNeasy Mini Kit (Qiagen). Genomic DNA was removed by on-column digestion using the RNase-free DNase Set (Qiagen). Data from HEK293DYG cells showed no significant overlap in the ligand-treated transcriptomes obtained from HEK293DAX cells.

Project description:The unfolded protein response (UPR) maintains endoplasmic reticulum (ER) proteostasis through the activation of transcription factors such as XBP1s and ATF6. The functional consequences of these transcription factors for ER proteostasis remain poorly defined. Here, we describe methodology that enables orthogonal, small molecule-mediated activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the same cell independent of stress. We employ transcriptomics and quantitative proteomics to evaluate ER proteostasis network remodeling owing to the XBP1s and/or ATF6 transcriptional programs. Furthermore, we demonstrate that the three ER proteostasis environments accessible by activating XBP1s and/or ATF6 differentially influence the folding, trafficking, and degradation of destabilized ER client proteins without globally affecting the endogenous proteome. Our data reveal how the ER proteostasis network is remodeled by the XBP1s and/or ATF6 transcriptional programs at the molecular level and demonstrate the potential for selectively restoring aberrant ER proteostasis of pathologic, destabilized proteins through arm-selective UPR-activation. The unfolded protein response adapts endoplasmic reticulum (ER) proteostasis via stress-responsive transcription factors including XBP1s and ATF6. Here, R. Luke Wiseman and colleagues implement technology for the orthogonal, ligand-dependent activation of XBP1s and/or ATF6 in a single cell. They characterize how XBP1s and/or ATF6 activation impacts ER proteostasis pathway composition and function. Adapted ER environments influence the proteostasis of destabilized protein variants without affecting the endogenous proteome. The work informs the development of proteostasis environment-adapting therapeutics for protein misfolding-related diseases. In order to activate both XBP1s and ATF6 in the same cell, we incorporated DHFR.ATF6 and tet-inducible XBP1s into a HEK293T-REx cell line stably expressing the tet-repressor. Selection of a single colony resulted in the HEK293DAX cell line in which XBP1s is induced by doxycycline and DHFR.ATF6 is activated by trimethoprim (TMP; TMP-dependent DHFR.ATF6 activation in HEK293DAX cells will henceforth be referred to as ATF6 activation for simplicity). HEK293DAX cells were treated for 12 h with vehicle, 1 ?g/mL dox, 10 ?M TMP, or both in biological triplicate. Cells were harvested and RNA was extracted using the RNeasy Mini Kit (Qiagen). Genomic DNA was removed by on-column digestion using the RNase-free DNase Set (Qiagen). Data from HEK293DYG cells showed no significant overlap in the ligand-treated transcriptomes obtained from the control HEK293DYG cells.

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Transcriptional stress responses induced by compartment-targeted misfolding-prone proteins in Drosophila

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