Biology subjects

Plate, L.

Publications and source records attributed to Plate, L..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Elexacaftor/VX-445-mediated CFTR interactome remodeling reveals differential correction driven by mutation-specific translational dynamics

Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as Lumacaftor (VX-809), Tezacaftor (VX-661) and Elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry (AP-MS) multiplexed with isobaric Tandem Mass Tags (TMT) was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knock-downs sensitize P67L to VX-445 and further enhance the correction of this variant. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize unresponsive CFTR variants to known and available correctors.

cell biology↗

bioRxiv2022

Elucidation of Global Trends in the Effects of VX-661 and VX-445 on the Expression of Clinical CFTR Variants

Cystic fibrosis (CF) is a chronic genetic disease caused by mutations that compromise the expression and/ or function of the cystic fibrosis transmembrane conductance regulator chloride channel (CFTR). Most people with CF harbor a common misfolded CFTR variant ({Delta}F508), which can be rescued by combination therapies containing "corrector" compounds that restore its expression. Nevertheless, there are over 400 other CF variants that differ in their sensitivity to correctors for reasons that remain unclear. In this work, we utilize deep mutational scanning to quantitatively compare the effects of two FDA-approved correctors on the plasma membrane expression of 129 known CF variants, including 45 that are currently unclassified. Across 67 variants with attenuated expression, we find that VX-661-sensitive variants generally exhibit intermediate expression and feature mutations near its binding pocket in transmembrane domains (TMDs) 1, 2, 3, and 6. VX-445 also primarily rescues variants with intermediate expression but is instead uniquely effective towards mutations near its binding pocket in TMDs 10 & 11. Structural calculations suggest corrector binding provides similar stabilization to both sensitive and insensitive variants. These findings collectively suggest the mutation-specific effects of these compounds depend on the degree of variant destabilization and/ or the timing of cotranslational folding defects. Combining these correctors synergistically rescues variants with deficient and intermediate expression alike, presumably by doubling the total binding energy and suppressing defects at different stages of translation. These results provide an unprecedented overview of the properties of rare CFTR variants and establish new tools for CF pharmacology.

biochemistry↗

bioRxiv2022

The histone chaperone NASP maintains H3-H4 reservoirs in the early Drosophila embryo

Histones are essential for chromatin packaging and histone supply must be tightly regulated as excess histones are toxic. To drive the rapid cell cycles of the early embryo, however, excess histones are maternally deposited. Therefore, soluble histones must be buffered by histone chaperones but the chaperone necessary to stabilize soluble H3-H4 pools in the Drosophila embryo has yet to be identified. Here, we show that CG8223, the Drosophila ortholog of NASP, is a H3-H4-specific chaperone in the early embryo. NASP specifically binds to H3-H4 in the early embryo. We demonstrate that, while a NASP null mutant is viable in Drosophila, NASP is maternal effect lethal gene. Embryos laid by NASP mutant mothers have a reduce rate of hatching and show defects in early embryogenesis. Critically, soluble H3-H4 pools are degraded in embryos laid by NASP mutant mothers. Our work identifies NASP as the critical H3-H4 histone chaperone in the Drosophila embryo.

developmental biology↗

bioRxiv2022

Time-Resolved Interactome Profiling Deconvolutes Secretory Protein Quality Control Dynamics

Many cellular processes are governed by protein-protein interactions that require tight spatial and temporal regulation. Accordingly, it is necessary to understand the dynamics of these interactions to fully comprehend and elucidate cellular processes and pathological disease states. To map de novo protein-protein interactions with time-resolution at an organelle-wide scale we developed a quantitative mass-spectrometry method, time-resolved interactome profiling (TRIP). We apply TRIP to elucidate aberrant protein interaction dynamics that lead to the protein misfolding disease congenital hypothyroidism. We deconvolute altered temporal interactions of the thyroid hormone precursor thyroglobulin with pathways implicated in hypothyroidism pathophysiology such as Hsp70/90 assisted folding, disulfide/redox processing, and N-glycosylation. Functional siRNA screening identified VCP and TEX264 as key protein degradation components whose inhibition selectively rescues mutant prohormone secretion. Ultimately, our results provide novel insight into the temporal coordination of protein homeostasis, and our TRIP method should find broad applications in investigating protein folding diseases and cellular processes.

cell biology↗

bioRxiv2022

The unfolded protein response transcription factor XBP1s ameliorates Alzheimer`s disease by improving synaptic function and proteostasis

Alteration in the buffering capacity of the proteostasis network is an emerging feature of Alzheimers disease (AD), highlighting the occurrence of endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is the main adaptive pathway to cope with protein folding stress at the ER. Inositol requiring enzyme-1 (IRE1) is an ER-located kinase and endoribonuclease that operates as a central ER stress sensor, enabling the establishment of adaptive and repair programs through the control of the expression of the transcription factor X-Box binding protein 1 (XBP1). A polymorphism in the XBP1 promoter has been suggested as a risk factor for AD. To artificially enforce the adaptive capacity of the UPR in the AD brain, we developed strategies to express the active form of XBP1 in neurons using preclinical models. Overexpression of an active form of XBP1 in the nervous system using transgenic mice significantly reduced the load of amyloid deposits in the cerebral cortex and hippocampus and preserved synaptic and cognitive function. Moreover, local delivery of XBP1 into the hippocampus of an AD mice using adeno-associated vectors improved long-term potentiation, memory performance, and dendritic spine density. Quantitative proteomics of the hippocampus revealed that XBP1 expression corrects a large proportion of the alterations observed in the 5xFAD model, restoring the levels of several synaptic proteins and factors involved in actin cytoskeleton regulation and axonal growth. Our results illustrate the therapeutic potential of targeting UPR-dependent gene expression programs as a strategy to ameliorate AD features and sustain synaptic function.

animal behavior and cognition↗

bioRxiv2022

Identification of replication fork-associated proteins in Drosophila embryos and cultured cells using iPOND coupled to quantitative mass spectrometry

Replication of the eukaryotic genome requires the formation of thousands of replication forks that must work in concert to accurately replicate the genetic and epigenetic information. Defining replication fork-associated proteins is a key step in understanding how genomes are replicated and repaired in the context of chromatin to maintain genome stability. To identify replication fork-associated proteins, we performed iPOND (Isolation of Proteins on Nascent DNA) coupled to quantitative mass spectrometry in Drosophila embryos and cultured cells. We identified 76 and 278 fork-associated proteins in post-MZT embryos and Drosophila cultured S2 cells, respectively. By performing a targeted screen of a subset of these proteins, we demonstrate that BRWD3, a targeting specificity factor for the DDB1/Cul4 ubiquitin ligase complex (CRL4), functions at replication forks to promote fork progression and maintain genome stability. Altogether, our work provides a valuable resource for those interested in the DNA replication, repair and chromatin assembly during development.

biochemistry↗

bioRxiv2021

Structural comparative modeling of multi-domain ΔF508 CFTR

Cystic Fibrosis (CF) is a common genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), an epithelial anion channel expressed in several vital organs. Absence of functional CFTR results in imbalanced osmotic equilibrium and subsequent mucus build up in the lungs - which increases the risk of infection and eventually causes death. CFTR is an ATP binding cassette (ABC) transporter composed of two transmembrane domains (TMDs), two nucleotide binding domains (NBDs), and an unstructured regulatory domain. The most prevalent patient mutation is the deletion of F508 ({Delta}F508), making {Delta}F508 CFTR the primary target for current FDA approved CF therapies. However, no experimental multi-domain {Delta}F508 CFTR structure has been determined and few studies have modeled {Delta}F508 using multi-domain WT CFTR structures. Here, we used cryo-EM density data and Rosetta comparative modeling (RosettaCM) to compare a {Delta}F508 model with published experimental data on CFTR NBD1 thermodynamics. We then apply this modeling method to generate multi-domain WT and {Delta}F508 CFTR structural models. These models demonstrate the destabilizing effects of {Delta}F508 on NBD1 and the NBD1/TMD interface in both the closed and open conformation of CFTR. Furthermore, we modeled {Delta}F508/R1070W and {Delta}F508 bound to the CFTR corrector VX-809. Our models reveal the stabilizing effects of R1070W and VX-809 on multi-domain models of {Delta}F508 CFTR and pave the way for rational design of additional drugs that target {Delta}F508 CFTR for treatment of CF. Author SummaryProteins three-dimension shape determines their function, so when genetic mutation compromises the shape of vital proteins, it may cause disease. Such is the case in Cystic Fibrosis, a chronic genetic disease caused by mutations in the protein Cystic Fibrosis Transmembrane Conductance Regulator. Here, we work backwards from the shape of the wild-type protein - found in healthy people, to computationally model the shape of the most common Cystic Fibrosis mutant. Our computer models reveal distinct defects in the shape of the mutant Cystic Fibrosis Transmembrane Conductance Regulator protein in the area surrounding the mutation. We also model an important FDA approved Cystic Fibrosis drug, VX-809, into the mutant protein structure and show how VX-809 stabilizes the protein around the location of the mutation. The method we developed will pave the way for computational drug design for Cystic Fibrosis.

bioinformatics↗

bioRxiv2021

Distinct proteostasis states drive pharmacologic chaperone susceptibility for Cystic Fibrosis Transmembrane Conductance Regulator misfolding mutants

Pharmacological chaperones represent a class of therapeutic compounds for treating protein misfolding diseases. One of the most prominent examples is the FDA-approved pharmacological chaperone lumacaftor (VX-809), which has transformed cystic fibrosis (CF) therapy. CF is a fatal disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). VX-809 corrects folding of F508del CFTR, the most common patient mutation, yet F508del exhibits only mild VX-809 response. In contrast, rarer mutations P67L and L206W are hyper-responsive to VX-809, while G85E is non-responsive. Despite the clinical success of VX-809, the mechanistic origin for the distinct susceptibility of mutants remains unclear. Here, we use interactomics to characterize the impact of VX-809 on proteostasis interactions of P67L and L206W and compare these to F508del and G85E. We determine hyper-responsive mutations P67L and L206W exhibit decreased interactions with proteasomal, and autophagy degradation machinery compared to F508del and G85E. We then show inhibiting the proteasome attenuates P67L and L206W VX-809 response, and inhibiting the lysosome attenuates F508del VX-809 response. Our data suggests a previously unidentified but required role for protein degradation in VX-809 correction. Furthermore, we present an approach for identifying proteostasis characteristics of mutant-specific therapeutic response to pharmacological chaperones.

cell biology↗

bioRxiv2021

A Family of Glycosylated Macrolides Selectively Target Energetic Vulnerabilities in Leukemia

Cancer cells have long been recognized to exhibit unique bioenergetic requirements. The apoptolidin family of glycomacrolides are distinguished by their selective cytotoxicity towards oncogene transformed cells, yet their molecular mechanism remains uncertain. We used photoaffinity analogs of the apoptolidins to identify the F1 subcomplex of mitochondrial ATP synthase as the target of apoptolidin A. CryoEM of apoptolidin and ammocidin-ATP synthase complexes revealed a novel shared mode of inhibition that was confirmed by deep mutational scanning of the binding interface to reveal resistance mutations which were confirmed using CRISPR-Cas9. Ammocidin A was found to suppress leukemia progression in vivo at doses that were tolerated with minimal toxicity. The combination of cellular, structural, mutagenesis, and in vivo evidence define the mechanism of action of apoptolidin family glycomacrolides and establish a path to address OXPHOS-dependent cancers.

biochemistry↗

bioRxiv2021

Comparative host interactomes of the SARS-CoV-2 nonstructural protein 3 and human coronavirus homologs

Human coronaviruses have become an increasing threat to global health; three highly pathogenic strains have emerged since the early 2000s, including most recently SARS-CoV-2, the cause of COVID-19. A better understanding of the molecular mechanisms of coronavirus pathogenesis is needed, including how these highly virulent strains differ from those that cause milder, common-cold like disease. While significant progress has been made in understanding how SARS-CoV-2 proteins interact with the host cell, non-structural protein 3 (nsp3) has largely been omitted from the analyses. Nsp3 is a viral protease with important roles in viral protein biogenesis, replication complex formation, and modulation of host ubiquitinylation and ISGylation. Herein, we use affinity purification-mass spectrometry to study the host-viral protein-protein interactome of nsp3 from five coronavirus strains: pathogenic strains SARS-CoV-2, SARS-CoV, and MERS-CoV; and endemic common-cold strains hCoV-229E and hCoV-OC43. We divide each nsp3 into three fragments and use tandem mass tag technology to directly compare the interactors across the five strains for each fragment. We find that few interactors are common across all variants for a particular fragment, but we identify shared patterns between select variants, such as ribosomal proteins enriched in the N-terminal fragment (nsp3.1) dataset for SARS-CoV-2 and SARS-CoV. We also identify unique biological processes enriched for individual homologs, for instance nuclear protein important for the middle fragment of hCoV-229E, as well as ribosome biogenesis of the MERS nsp3.2 homolog. Lastly, we further investigate the interaction of the SARS-CoV-2 nsp3 N-terminal fragment with ATF6, a regulator of the unfolded protein response. We show that SARS-CoV-2 nsp3.1 directly binds to ATF6 and can suppress the ATF6 stress response. Characterizing the host interactions of nsp3 widens our understanding of how coronaviruses co-opt cellular pathways and presents new avenues for host-targeted antiviral therapeutics.

biochemistry↗

bioRxiv2020

Glycosylation Limits Forward Trafficking of the Tetraspan Membrane Protein PMP22

Peripheral myelin protein 22 (PMP22) folds and traffics inefficiently, a phenomenon closely related to the mechanisms by which this tetraspan membrane protein causes Charcot-Marie-Tooth disease (CMTD). We report that elimination of N-glycosylation results in a 3-fold increase in the cell surface trafficking of wild type (WT) PMP22 and a 10-fold increase in trafficking of the unstable L16P disease mutant form. Studies of the interactions of PMP22 with oligosaccharyltransferases A and B as well as quantitative proteomic experiments established that critical endoplasmic reticulum (ER) quality control decisions occur earlier in the biogenesis to cell surface trafficking pathway for the L16P mutant than for WT. CRISPR knock-out cell lines for ER proteins calnexin, RER1, and UGGT1 illuminated the role of each protein in glycosylation dependent and independent surface trafficking of WT PMP22, as well as for a series of disease mutants of varying folding stabilities. One Sentence SummaryN-linked glycosylation was seen to dramatically limit the cell surface trafficking of PMP22, with some key quality control factors in PMP22 biogenesis being identified.

cell biology↗

bioRxiv2020

Enforced dimerization between XBP1s and ATF6f enhances the protective effects of the unfolded protein response (UPR) in models of neurodegeneration

Alteration to endoplasmic reticulum (ER) proteostasis is observed on a variety of neurodegenerative diseases associated with abnormal protein aggregation. Activation of the unfolded protein response (UPR) enables an adaptive reaction to recover ER proteostasis and cell function. The UPR is initiated by specialized stress sensors that engage gene expression programs through the concerted action of the transcription factors ATF4, ATF6f, and XBP1s. Although UPR signaling is generally studied as unique linear signaling branches, correlative evidence suggests that ATF6f and XBP1s may physically interact to regulate a subset of UPR-target genes. Here, we designed an ATF6f-XBP1s fusion protein termed UPRplus that behaves as a heterodimer in terms of its selective transcriptional activity. Cell-based studies demonstrated that UPRplus has stronger an effect in reducing the abnormal aggregation of mutant huntingtin and alpha-synuclein when compared to XBP1s or ATF6 alone. We developed a gene transfer approach to deliver UPRplus into the brain using adeno-associated viruses (AAVs) and demonstrated potent neuroprotection in vivo in preclinical models of Parkinsons and Huntingtons disease. These results support the concept where directing UPR-mediated gene expression toward specific adaptive programs may serve as a possible strategy to optimize the beneficial effects of the pathway in different disease conditions.

neuroscience↗

bioRxiv2020

Comparative multiplexed interactomics of SARS-CoV-2 and homologous coronavirus non-structural proteins identifies unique and shared host-cell dependencies

Human coronaviruses (hCoV) have become a threat to global health and society, as evident from the SARS outbreak in 2002 caused by SARS-CoV-1 and the most recent COVID-19 pandemic caused by SARS-CoV-2. Despite high sequence similarity between SARS-CoV-1 and -2, each strain has distinctive virulence. A better understanding of the basic molecular mechanisms mediating changes in virulence is needed. Here, we profile the virus-host protein-protein interactions of two hCoV non-structural proteins (nsps) that are critical for virus replication. We use tandem mass tag-multiplexed quantitative proteomics to sensitively compare and contrast the interactomes of nsp2 and nsp4 from three betacoronavirus strains: SARS-CoV-1, SARS-CoV-2, and hCoV-OC43 - an endemic strain associated with the common cold. This approach enables the identification of both unique and shared host cell protein binding partners and the ability to further compare the enrichment of common interactions across homologs from related strains. We identify common nsp2 interactors involved in endoplasmic reticulum (ER) Ca2+ signaling and mitochondria biogenesis. We also identifiy nsp4 interactors unique to each strain, such as E3 ubiquitin ligase complexes for SARS-CoV-1 and ER homeostasis factors for SARS-CoV-2. Common nsp4 interactors include N-linked glycosylation machinery, unfolded protein response (UPR) associated proteins, and anti-viral innate immune signaling factors. Both nsp2 and nsp4 interactors are strongly enriched in proteins localized at mitochondrial-associated ER membranes suggesting a new functional role for modulating host processes, such as calcium homeostasis, at these organelle contact sites. Our results shed light on the role these hCoV proteins play in the infection cycle, as well as host factors that may mediate the divergent pathogenesis of OC43 from SARS strains. Our mass spectrometry workflow enables rapid and robust comparisons of multiple bait proteins, which can be applied to additional viral proteins. Furthermore, the identified common interactions may present new targets for exploration by host-directed anti-viral therapeutics.

biochemistry↗

bioRxiv2020

Insulin-like growth factor 2 (IGF2) protects against Huntington's disease through the extracellular disposal of protein aggregates

Impaired neuronal proteostasis is a salient feature of many neurodegenerative diseases, highlighting alterations in the function of the endoplasmic reticulum (ER). We previously reported that targeting the transcription factor XBP1, a key mediator of the ER stress response, delays disease progression and reduces protein aggregation in various models of neurodegeneration. To identify disease-modifier genes that may explain the neuroprotective effects of XBP1 deficiency, we performed gene expression profiling of brain cortex and striatum of these animals and uncovered insulin-like growth factor 2 (Igf2) as the major upregulated gene. Here we studied the impact of IGF2 signaling on protein aggregation in models of Huntingtons disease (HD) as proof-of-concept. Cell culture studies revealed that IGF2 treatment decreases the load of intracellular aggregates of mutant huntingtin and a polyglutamine peptide. These results were validated using induced pluripotent stem cells (iPSC)-derived medium spiny neurons from HD patients. The reduction in the levels of mutant huntingtin was associated with a decrease in the half-life of the intracellular protein. The decrease in the levels of abnormal protein aggregation triggered by IGF2 were independent of the activity of autophagy and the proteasome pathways, the two main routes for mutant huntingtin clearance. Conversely, IGF2 signaling enhanced the secretion of soluble mutant huntingtin species through exosomes and microvesicles involving changes in actin dynamics. Administration of IGF2 into the brain of HD mice using gene therapy led to a significant decrease in the levels of mutant huntingtin in three different animal models. Moreover, analysis of human post-mortem brain tissue, and blood samples from HD patients showed a reduction of IGF2 level. This study identifies IGF2 as a relevant factor deregulated in HD, operating as a disease modifier that buffers the accumulation of abnormal protein aggregates. One sentence summaryIGF2 reduces the load of intracellular protein aggregates through the extracellular disposal of the mutant protein.

neuroscience↗

bioRxiv2020

Dengue and Zika virus infection are impaired by small molecule ER proteostasis regulator 147 in an ATF6-independent manner

Flaviviruses, including Dengue and Zika, are widespread human pathogens, however, no broadly active therapeutics exist to fight infection. Here, we establish the recently discovered pharmacologic modulator of ER proteostasis 147 as an effective host-centered antiviral strategy. Compound 147 reduces infection by attenuating viral replication without causing toxicity in host cells. 147 is a preferential activator of the ATF6 pathway of the unfolded protein response, which requires targeting of cysteine residues primarily on protein disulfide isomerases (PDIs). We find that the antiviral activity of 147 is independent of ATF6 induction but does require modification of reactive thiols on protein targets. Targeting PDIs using RNAi and other PDI small molecule inhibitors was unable to recapitulate the antiviral effects, suggesting additional identified protein targets of 147 may mediate the activity. Importantly, 147 can impair infection of multiple strains of Dengue and Zika virus, indicating that it is suitable as a broad-spectrum antiviral agent.

biochemistry↗

bioRxiv2020

Control of mammalian brain aging by the unfolded protein response (UPR)

Aging is the major risk factor for the development of dementia and neurodegenerative disorders, and the aging brain manifests severe deficits in buffering capacity by the proteostasis network. Accordingly, we investigated the significance of the unfolded protein response (UPR), a major signaling pathway that copes with endoplasmic reticulum (ER) stress, to normal mammalian brain aging. Genetic disruption of ER stress sensor IRE1 accelerated cognitive and motor dysfunction during aging. Exogenous bolstering of the UPR by overexpressing an active form of the transcription factor XBP1 restored synaptic and cognitive function in addition to reducing cell senescence. Remarkably, proteomic profiling of hippocampal tissue indicated that XBP1s expression corrected age-related alterations in synaptic function. Collectively, our data demonstrate that strategies to manipulate the UPR sustain healthy brain aging. One Sentence SummaryThe IRE1/XBP1 pathway dictates when and how brain function declines during aging.

neuroscience↗

bioRxiv2020

Thyroglobulin Interactome Profiling Uncovers Molecular Mechanisms of Thyroid Dyshormonogenesis

Thyroglobulin (Tg) is a secreted iodoglycoprotein serving as the precursor for T3 and T4 hormones. Many characterized Tg gene mutations produce secretion-defective variants resulting in congenital hypothyroidism (CH). Tg processing and secretion is controlled by extensive interactions with chaperone, trafficking, and degradation factors comprising the secretory proteostasis network. While dependencies on individual proteostasis network components are known, the integration of proteostasis pathways mediating Tg protein quality control and the molecular basis of mutant Tg misprocessing remain poorly understood. We employ a multiplexed quantitative affinity purification-mass spectrometry approach to define the Tg proteostasis interactome and changes between WT and several CH-variants. Mutant Tg processing is associated with common imbalances in proteostasis engagement including increased chaperoning, oxidative folding, and routing towards ER-associated degradation components, yet variants are inefficiently degraded. Furthermore, we reveal mutation-specific changes in engagement with N-glycosylation components, suggesting distinct requirements for one Tg variant on dual engagement of both oligosaccharyltransferase complex isoforms for degradation. Modulating dysregulated proteostasis components and pathways may serve as a therapeutic strategy to restore Tg secretion and thyroid hormone biosynthesis.

cell biology↗

bioRxiv2018

Deconvoluting Stress-Responsive Proteostasis Signaling Pathways for Pharmacologic Activation using Targeted RNA-sequencing

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 stress-responsive 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 stress-responsive 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.

cell biology↗