Deconvoluting Stress-Responsive Proteostasis Signaling Pathways for Pharmacologic Activation Using Targeted RNA Sequencing

Abstract

Cellular proteostasis is maintained by stress-responsive signaling pathways such as the heat shock response (HSR), the oxidative stress response (OSR), and the unfolded protein response (UPR). Activation of these pathways results in the transcriptional upregulation of select subsets of stressresponsive genes that restore proteostasis and adapt cellular physiology to promote recovery following various types of acute insult. The capacity for these pathways to regulate cellular proteostasis makes them attractive therapeutic targets to correct proteostasis defects associated with diverse diseases. High-throughput screening (HTS) using cell-based reporter assays is highly effective for identifying putative activators of stress-responsive signaling pathways. However, the development of these compounds is hampered by the lack of medium-throughput assays to define compound potency and selectivity for a given pathway. Here, we describe a targeted RNA sequencing (RNAseq) assay that allows cost effective, medium-throughput screening of stressresponsive signaling pathway activation. We demonstrate that this assay allows deconvolution of stress-responsive signaling activated by chemical genetic or pharmacologic agents. Furthermore, we use this assay to define the selectivity of putative OSR and HSR activating compounds previously identified by HTS. Our results demonstrate the potential for integrating this adaptable targeted RNAseq assay into screening programs focused on developing pharmacologic activators of stress-responsive signaling pathways.

Acute unfolding of a single protein immediately stimulates recruitment of ubiquitin protein ligase E3C (UBE3C) to 26S proteasomes

“…Much of our understanding of cellular protein homeostasis networks comes from studies in which cells are subjected to insults like heat shock (35), Hsp90 inhibitors (36), or chemical denaturants or oxidants (37,38) that cause immediate disruption to global protein conformation. Although such approaches have been instrumental in revealing the existence of homeostatic networks that promote stereotypical, temporally orchestrated restoration of homeostasis, animal cells are rarely, if ever, subjected to such acute, massive, proteome-wide insults.…”

Defining the Functional Targets of Cap‘n’collar Transcription Factors NRF1, NRF2, and NRF3

“…While recent research has spelled out important roles for NRF1 and NRF2 in regulating cellular physiology, the functional importance for three stress-inducible CNC-containing transcription factors with overlapping, but distinct, transcriptional profiles in vertebrates is not completely defined. One approach to deconvolute the diverse functional roles for these transcription factors is to define transcriptional gene sets that can be used to define transcription factor activity both in cell culture and mammalian tissues [ 18 ]. To date, only one recent study by Liu et al has aimed to comparatively delineate the transcriptional programs enacted by NRF factors [ 19 ].…”

Multiexon deletion alleles of ATF6 linked to achromatopsia

“…HEK293 cells expressing WT or mutant ATF6 were collected for RNA-Seq analysis. Cells were lysed and total RNA was collected using a Qiagen kit (RNeasy Mini Kit) in line with the manufacturer's recommendations and previous study (49). Three sets of independent experiments were performed.…”