Stress-Independent Activation of XBP1s and/or ATF6 Reveals Three Functionally Diverse ER Proteostasis Environments

Abstract

Summary 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 selective restoration of aberrant ER proteostasis of pathologic, destabilized proteins through arm-selective UPR activation.

Unfolded protein response activation reduces secretion and extracellular aggregation of amyloidogenic immunoglobulin light chain

“…Reduced FT ALLC secretion induced by XBP1s or ATF6 activation was dependent on transcription factor activity, as no reduction in FT ALLC secretion was observed in TMP-or Dox-treated HEK293 DYG cells-a control cell line stably expressing DHFR-YFP and Dox-inducible GFP (Fig. S4B) (40).…”

“…Previous work has indicated that remodeling of ER proteostasis pathways selectively influences the secretion of destabilized protein variants relative to more stable protein variants (13,40,41). Thus, Tg-dependent UPR activation could selectively decrease the secretion of destabilized ALLC through the transcriptional remodeling of the ER proteostasis network.…”

“…In these cells, the ATF6 or XBP1s transcriptional programs can be orthogonally controlled in the absence of ER stress, which enables analysis of the functional consequences of arm-selective UPR activation independently, or in combination (Fig. S4A) (40).…”

“…Thus, Tg-dependent UPR activation could selectively decrease the secretion of destabilized ALLC through the transcriptional remodeling of the ER proteostasis network. To differentiate between the potential effects of Tg-induced ER stress and UPR-dependent remodeling of ER proteostasis pathways on ALLC secretion, we used a HEK293T-Rex-derived cell line that expresses both a ligand-regulatable ATF6 transcription factor [DHFR-ATF6; activated by the addition of the small molecule pharmacologic chaperone trimethoprim (TMP)] and a doxycycline (Dox)-inducible XBP1s transcription factor (cells referred to as HEK293 DAX ) (40). In these cells, the ATF6 or XBP1s transcriptional programs can be orthogonally controlled in the absence of ER stress, which enables analysis of the functional consequences of arm-selective UPR activation independently, or in combination (Fig.…”

Multiple Mechanisms of Unfolded Protein Response–Induced Cell Death

“…18,19 The cytosolic portion of ATF6 contains the bZIP transcriptional activator domain, and after cleavage, this ATF6 fragment migrates to the nucleus to transcriptionally up-regulate ER chaperones and ERAD components, thereby enhancing ER proteinfolding capacity and efficiency of ERAD. 10,12,18,20 Interestingly, ATF6 also transcriptionally up-regulates Xbp1, thereby facilitating IRE1 signal transduction by increasing levels of IRE1's RNase substrate, Xbp1 mRNA. 21 Put together, these initial transcriptional and translational effects of IRE1, PERK, and ATF6 signaling help cells adapt to ER stress by enhancing the fidelity of protein folding, increasing the degradation of damaged/misfolded proteins, and suppressing new protein synthesis.…”