TUDCA modulates drug bioavailability to regulate resistance to acute ER stress in Saccharomyces cerevisiae

INTRODUCTION

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) results in ER stress that activates several response pathways to restore homeostasis of the secretory protein folding environment. In particular, the Unfolded Protein Response (UPR) responds to both accumulation of misfolded secretory proteins in the ER lumen, as well as ER membrane lipid perturbations (Gardner and Walter, 2011; Lajoie et al., 2012; Volmer et al., 2013; Volmer and Ron, 2015; Halbleib et al., 2017; Karagöz et al., 2017; Fun and Thibault, 2020; Ho et al., 2020; Ishiwata-Kimata et al., 2021; Celik et al., 2023). UPR sensors activate a transcriptional response to adapt cells to restore homeostasis (Harding et al., 2000; Travers et al., 2000; Walter and Ron, 2011).

ER stress is a feature of numerous conditions, including neurodegenerative diseases (e.g., Alzheimer's and Parkinson's Diseases), chronic conditions (e.g., diabetes and inflammation), as well as the aging process (Jiang et al., 2016; García-González et al., 2018; Chadwick and Lajoie, 2019; Taylor and Hetz, 2020; Ren et al., 2021). Chronic unresolved ER stress can lead to cell death (Chawla et al., 2011; Rubio et al., 2011; Hetz and Papa, 2018; Rai et al., 2024) and exacerbate disease pathology. Modulation of ER stress has been proposed as a strategy to reduce the severity of associated diseases. Several pharmacologic compounds have been developed that selectively up-regulate and down-regulate arms of the UPR (Maly and Papa, 2014; Gallagher and Walter, 2016; Gonzalez-Teuber et al., 2019; Hetz et al., 2019a; Marciniak et al., 2022). In a parallel approach, ER stress could (theoretically) be ameliorated by directly decreasing the misfolded secretory protein burden either by refolding, degrading, or decreasing the synthesis of misfolded/unfolded proteins (Hetz et al., 2019b; Grandjean and Wiseman, 2020; Kelly, 2020). A broad group of small molecules appear to improve the secretory protein folding environment by possibly directly assisting protein folding, stability, or trafficking. These compounds have been termed “chemical chaperones” (Welch and Randell Brown, 1996; Papp and Csermely, 2006). Importantly, the different reported chemical chaperones appear to have distinct impacts on protein folding capacity and stress coping mechanisms (Uppala et al., 2017).

Tauroursodeoxycholic acid (TUDCA) has been described as a chemical chaperone and shows promise in therapeutically decreasing ER stress. TUDCA is a taurine-conjugated bile acid produced in small amounts in the human body by intestinal bacteria (Winston and Theriot, 2020). It is the primary bile acid produced in Asian and North American black bears (Wang and Carey, 2014; Li et al., 2016). TUDCA (and bear bile acid in general) has been used for centuries as a traditional Chinese remedy (Qiao et al., 2011) and can improve symptoms and slow progression of numerous ER stress–associated diseases including neurodegeneration, cardiac dysfunction, retinal degeneration, and type 2 diabetes (Keene et al., 2002; Ozcan et al., 2006; Rivard et al., 2007; Boatright et al., 2009; Berger and Haller, 2011; Nunes et al., 2012; Lawson et al., 2016; Lojpur et al., 2019). TUDCA reportedly improves secretory protein folding diseases and attenuates UPR activation (Xie et al., 2002; Ozcan et al., 2006; Cao et al., 2013; Uppala et al., 2017). TUDCA has been hypothesized to decrease UPR activation by stabilizing protein conformation, thereby improving the folding capacity of the ER and decreasing ER stress (Ozcan et al., 2006; Omura et al., 2013). Due to the interest in TUDCA as a treatment for multiple human diseases, determining the mechanism of how TUDCA might regulate ER stress could identify the best targets for TUDCA, as well as suggesting routes to modify or enhance TUDCA activity (Kusaczuk, 2019).

To investigate the protective mechanism of TUDCA at the cellular level, we leveraged the genetically tractable budding yeast Saccharomyces cerevisiae. The UPR is evolutionarily ancient. While metazoans have a more complex response with five stress UPR sensors (IRE1ɑ and β, PERK and ATF6ɑ and β) (Walter and Ron, 2011; Wu et al., 2014), yeast have only the highly conserved Ire1 UPR sensor (Walter and Ron, 2011; Wu et al., 2014). In yeast, Ire1 cleaves the mRNA of the XBP1 homologue HAC1, which is then spliced into a shorter form that stimulates transcription of ∼400 genes involved in secretory protein folding, degradation, trafficking, and lipid synthesis (Cox et al., 1993; Cox and Walter, 1996; Shamu and Walter, 1996; Travers et al., 2000). Previous studies of proposed chemical chaperones (such as 4-PBA, DMSO, TMAO, and glycerol) have employed yeast models to assess the impact of these drugs on protein folding and trafficking, as well as downstream effects on conserved protein quality control machinery and pathways (Singh et al., 2007; Mai et al., 2018, 2019). However, several of these compounds are now considered proteostasis modulators that can either readjust protein folding and trafficking efficiency and/or directly regulate proteostasis by acting on specific stress response pathways such as the UPR (Balch et al., 2008; Ma et al., 2017). For example, treatment with 4-PBA can downregulate the UPR induced by tunicamycin (Tm) in yeast (Ho et al., 2020). Specifically, 4-PBA increases misfolded secretory protein sorting into COPII vesicles and thus decreases levels of misfolded proteins in the ER lumen (Ma et al., 2017). By using yeast genetics and cell imaging assays, we sought to determine the mechanism by which TUDCA could resolve different forms of ER stress. Our study reveals a surprising mode of action for TUDCA and suggests TUDCA should no longer be classified as a chemical chaperone.

RESULTS

TUDCA restores growth in presence of Tm

We set out to systematically define the mode(s) of action by which TUDCA could decrease ER stress. We began by considering the possibilities that TUDCA could act by enhancing the intensity of the ER stress response, accelerating resolution of the stress response, decreasing the burden of unfolded secretory proteins, decreasing stressor levels in the cell, and/or improving cell function to counterbalance the effects of misfolded protein stress. Before dissecting mechanisms, we began with a simple readout assay. ER stress can slow or inhibit cell growth and ultimately kill cells. Therefore, we asked whether TUDCA could improve yeast cell growth in the face of misfolded secretory protein stress. Yeast cells were spotted on agar plates containing the classic ER stressor Tm (an inhibitor of secretory protein N-glycosylation) (Kuo and Lampen, 1974). N-glycosylation can increase solubility of hydrophobic domains of proteins, enable binding of ER lectin chaperones, and ensure proper trafficking along the secretory pathway (Drickamer, 1988; Varki, 2017). Inhibition of N-glycosylation results in UPR activation (Rose et al., 1989; Cox and Walter, 1996). We tested a range of TUDCA concentrations for their ability to rescue Tm-induced growth defects of wild-type cells. Only 5 mM TUDCA restored growth (Figure 1A). Unless otherwise indicated, 5 mM TUDCA was used for subsequent experiments. If TUDCA improves protein folding, we reasoned TUDCA should decrease UPR activation upon Tm treatment. To measure UPR activation, cells expressing the fluorescent UPR reporter, UPR-mCherry (Merksamer et al., 2008), were treated with 2.5 µg/ml Tm ± TUDCA and assayed for UPR-mCherry expression over time (30–120 min) by flow cytometry (Figure 1B). As expected, UPR reporter expression increased with Tm treatment. However, at early times, cells cotreated with 5 mM TUDCA showed significantly lower UPR activation compared with cells treated with Tm alone. No significant difference in intensity was observed when the fluorescent protein reporter was expressed from a control UPR-independent GPD promoter (Figure 1C). Together, these results suggest TUDCA might improve growth by decreasing misfolded protein accumulation or otherwise improving the ER folding environment during Tm treatment.

TUDCA alleviates phenotypes associated with acute Tm treatment

Next, we investigated TUDCA's ability to modulate ER stress–associated phenotypes. We were interested in how broadly TUDCA rescues cells from a range of consequences of ER stress. For example, during acute ER stress induced by Tm or other stressors, small secretory proteins are retrotranslocated from the ER lumen to the cytoplasm, a phenomenon termed ER reflux (Igbaria et al., 2019; Lajoie and Snapp, 2020). Reflux can be observed in living cells by assessing the localization of a normally ER-localized GFP reporter, which relocalizes from the ER to the cytosol during misfolded protein stress (Igbaria et al., 2019; Lajoie and Snapp, 2020). Cells treated with Tm exhibited a reflux redistribution of fluorescence to the cytosol, but the reporter relocalization is attenuated in cells cotreated with TUDCA (Figure 2A). Similarly, TUDCA treatment significantly reduced other downstream consequences of Tm-induced ER stress, such as the attenuation of PKA activity (Pincus et al., 2014) (Figure 2B) and the relocalization of the transcription factor Sfp1 to the cytoplasm from the nucleus (Marion et al., 2004) (Figure 2, C and D).

Because Tm blocks N-linked glycosylation (causing a buildup of immature glycoproteins in the ER) (Kuo and Lampen, 1974), we assessed changes in the proportion of glycosylated species of an abundant glycoprotein reporter, Pdi1 (protein disulfide isomerase 1) (Tachikawa et al., 1991) by immunoblot. Pdi1 has five glycosylation sites, which significantly increase its mature molecular size. Impaired glycosylation is visible with an increase in the amount of nonglycosylated precursor species (lower molecular weight bands) and a decrease in the amount of the fully-glycosylated species (higher molecular weight bands). However, TUDCA did not restore glycosylation in the presence of high concentrations of Tm (1.0–2.5 µg/ml) (Figure 3A). Not all glycoproteins misfold if they fail to be glycosylated. To more broadly assess levels of misfolded secretory protein accumulation in the ER, we assayed changes in diffusion of the GFP-tagged Kar2 chaperone by fluorescence recovery after photobleaching (FRAP) (Figure 3, B and C). The diffusion coefficient of a molecule is inversely proportional to molecular size (Einstein, 1905). Larger molecules or molecular complexes diffuse more slowly than smaller molecules. Decreased mobility of Kar2 (BiP/GRP78 in mammalian cells) directly reflects its binding to misfolded proteins (Lai et al., 2010; Lajoie and Snapp, 2011; Lajoie et al., 2012). TUDCA treatment failed to rescue low Kar2 mobility during Tm treatment. Thus, TUDCA does not prevent Tm-induced accumulation of misfolded secretory proteins.

TUDCA differentially impacts ER stressors

Impressed with the ability of TUDCA to rescue cell growth in Tm, we were curious whether TUDCA could protect against other classes of ER stressors that induce protein misfolding. Cells cannot grow in another common UPR inducer DTT, a reducing agent that prevents disulfide bond formation and acutely increases misfolded protein accumulation (Tatu et al., 1993). Treatment with TUDCA failed to restore growth of cells in liquid cultures treated with DTT (Figure 4A). We next asked whether TUDCA can rescue growth of cells experiencing chronic, unmitigated UPR activation, known to be toxic to yeast cells (Chawla et al., 2011; Rubio et al., 2011). TUDCA did not improve, and appeared to further impair growth of cells expressing the constitutively active version (spliced) of Hac1 (HAC1i) (Figure 4B). Taken together with the results in Figure 1B, TUDCA does not appear to prevent UPR activation by another pharmacologic stressor or temper the negative consequences of UPR activation.

Next, we considered whether TUDCA could prevent or attenuate UPR induced by a single misfolded protein species. We reasoned that perhaps DTT might be too catastrophic of a stressor to correct. Therefore, we overexpressed a well-characterized misfolded mutant secretory protein. A single point mutation in the vacuolar carboxypeptidase Y (CPY) referred as CPY*, leads to its misfolding, retention, aggregation within the ER, UPR activation, and subsequent degradation via ERAD (Finger et al., 1993; Spear and Ng, 2003). Treating cells with TUDCA for up to 24 h failed to correct CPY* defects. CPY* still did not traffic out of the ER into the vacuole (Figure 4C). Nor did TUDCA treatment prevent CPY*-induced UPR activation. Indeed, prolonged TUDCA treatment exacerbated ER stress associated with CPY* and even with wild-type CPY overexpression (Figure 4D). Finally, we evaluated the impact of TUDCA on an entirely different category of ER stress, the stress that arises from the deletion of specific genes (Jonikas et al., 2009). We asked whether TUDCA treatment could attenuate stress in several deletion mutant strains with the UPR-mCherry reporter. spf1Δ cells (lack an ER ion transporter/ATPase) (Cronin et al., 2002), scj1Δ cells (lack an ER Hsp40 chaperone protein) (Silberstein et al., 1998), and ccw12Δ cells (lack a cell wall mannoprotein, which results in sensitivity to cell wall and osmotic stress) (Ragni et al., 2011) were treated with TUDCA for up to 24 h (Figure 4E). TUDCA did not alter UPR-mCherry expression in any of these cells. Thus, the benefits of TUDCA treatment appear to be limited.

Identification of genes required for Tm stress mitigation by TUDCA

Together, our results led us to hypothesize that TUDCA enabled cells to overcome stress associated with the action of Tm. Therefore, we narrowed our focus to identifying the molecular mechanism of how TUDCA rescued cell growth from the effects of Tm.

In an attempt to identify genes required for TUDCA's action, we screened the yeast nonessential deletion library (Giaever et al., 2002) for deletions that modulate the rescue of Tm-induced growth defects by TUDCA (Figure 5A). In accordance with our results that a functional UPR is still required for TUDCA to rescue growth at high Tm concentrations (Figure 1), UPR genes were identified in this screen (IRE1, HAC1). Interestingly, several genes required for TUDCA rescue of Tm treatment were related to other stress responses, including SLT2, MID2, BCK1, and RLM1, which are part of the cell wall integrity pathway and response to cell wall stress, HOG1 and PTC1, which regulate the response to osmotic stress, and CNB1 which regulates the calcineurin pathway (Figure 5B; Supplemental File S1). When analyzed by functional properties using TheCellMap (Costanzo et al., 2016; Usaj et al., 2017), the majority of gene deletion mutants treated with Tm, but not rescued by TUDCA were involved in glycosylation, protein folding, and the cell wall (Figure 5C). Thus, our screen data reveal that TUDCA does not replace the need for stress responses required to cope with Tm-induced stress, including the UPR.

TUDCA mitigates Tm-induced growth defect independently of the UPR

If TUDCA does not prevent gross accumulation of unglycosylated proteins or activation of the UPR, we asked whether TUDCA might act more subtly. To explore TUDCA's mechanism of action, we examined TUDCA's effects during treatment with low levels of Tm. Fortunately, Tm effects are titratable over concentrations of more than an order of magnitude. Even low drug concentrations partially inhibit N-glycosylation and activate detectable UPR (Rutkowski et al., 2006; Lajoie et al., 2012). We used the Pdi1 glycosylation assay to measure Tm activity and found that even 0.1 µg/ml Tm was sufficient to inhibit N-glycosylation of the majority of Pdi1 within 2 h (Figure 6A). However, at these lower Tm concentrations (0.1–0.5 µg/ml), TUDCA now prevented Pdi1 deglycosylation (Figure 6, A and B). In agreement with less accumulation of misfolded protein, we also observed less Tm-induced UPR in yeast expressing the UPR-mCherry reporter (Figure 6C). No change in fluorescence was observed for yeast constitutively expressing the GPD-driven yemRFP negative control (Figure 6D). Interestingly, these data suggest that TUDCA increases the concentration of Tm required to induce UPR. A direct prediction of this hypothesis is that TUDCA treatment could reduce the need for a functioning UPR to survive normally lethal low doses of Tm. Cells without a functional UPR (ire1Δ and hac1Δ) are especially sensitive to ER stress (Cox and Walter, 1996; Babour et al., 2010) and unable to grow on Tm plates, even at low concentrations. Indeed, we found that TUDCA could lower Tm toxicity sufficiently to reduce the requirement for UPR genes to enable yeast growth at a lower concentration (0.25 µg/ml) of Tm, but not 2-fold higher at 0.5 µg/ml (Figure 7). Thus, TUDCA can improve cell resistance to Tm independently of the UPR. Together, those data suggest that at lower concentrations of Tm, TUDCA decreases the effects of Tm to allow adaptation overtime and growth.

In light of the results from Figures 6 and 7, we revisited our findings in Figure 1. We hypothesized that TUDCA might protect cells from high doses of Tm by decreasing the effective Tm dose experienced by the cells and/or by accelerating the rate of adaptation to Tm stress. To test the latter hypothesis, we treated yeast expressing the UPR-mCherry reporter with 2.5 µg/ml Tm ± TUDCA and then measured mean reporter intensity at several timepoints. By 240 min, the TUDCA + Tm cells experienced significantly less stress than cells treated with Tm alone (Figure 8A). We measured the total area under the curve (Figure 8B) and further reinforced the interpretation that TUDCA decreased the total stress experienced. The UPR-mCherry reporter is not turned over and is a measure of total stress experienced, but does not indicate current stress level. To assess whether the rate of stress resolution changed with TUDCA treatment, we directly assayed splicing of the UPR effector, HAC1 mRNA by activated Ire1 (Figure 8C). At 2 h, similar levels of HAC1 splicing were observed in response to Tm with or without TUDCA. Yet, at 6 h, HAC1 splicing had decreased substantially in the presence versus the absence of TUDCA. In contrast, without TUDCA, Tm-induced HAC1 splicing only began to dissipate after 6 h. Thus, TUDCA decreased the total stress experienced and accelerated stress resolution.

Acute TUDCA treatment is not associated with profound transcriptional changes

We next considered the hypothesis that TUDCA reprograms cells to resist Tm. We complemented our genetics screen with a transcriptional profile of cells treated with TUDCA for 2 h, a timepoint when TUDCA treatment decreases UPR activation in cells treated with low doses of Tm (Figure 6C). We anticipated that a TUDCA protective stress response would be apparent by this time. Unexpectedly, TUDCA had only a modest effect on the yeast transcriptome. We identified 30 up-regulated and 29 down-regulated genes (adjusted p-value < 0.05) following TUDCA treatment (Supplemental Figure S1; Supplemental File S2). Differentially expressed genes were enriched for components of the cell periphery (cell wall/plasma membrane) and for active drug transporters (Supplemental Figure S1B). The plasma membrane ATP-binding cassette (ABC) transporter genes (PDR5, YOR1, SNQ2) involved in multidrug resistance were up-regulated. Interestingly, overexpression of PDR5 has been previously reported to alleviate pharmacologically-induced ER stress in yeast (Schmidt et al., 2019). We hypothesized that the transporters could be up-regulated by TUDCA to stimulate efflux of Tm. However, a mutant strain with all three transporters deleted only modestly decreased the ability of TUDCA to rescue the Tm-induced growth defect compared with the wild-type (Supplemental Figure S2). The difference may be unrelated to TUDCA effects as the transporter mutant strain has increased sensitivity to Tm (Rogers et al., 2001). Thus, the results do not support a model of TUDCA protection from Tm by multidrug transporter upregulation. Interestingly, differentially expressed genes were enriched for the Gene Ontology (GO) oxidoreductase activity category; TUDCA has been previously reported to regulate oxidative stress in other organisms (Wei et al., 2008; Cremers et al., 2014; Zhang and Wang, 2018; Hou et al., 2021; Pioltine et al., 2021). Thus, we tested the ability of TUDCA to alleviate oxidative stress. It was previously reported that a conserved cysteine within the ER chaperone Kar2 plays a critical role in sensing oxidative stress, and that mutation of Kar2’s Cys63 to an alanine residue confers sensitivity to diamide (Wang et al., 2014; Xu et al., 2016). TUDCA treatment improved the growth of cells expressing wild-type KAR2 and the kar2 C63A mutation in the presence of diamide (Supplemental Figure S3A). However, TUDCA did not rescue cells treated with hydrogen peroxide (Supplemental Figure S3B). Diamide causes up-regulation of genes associated with the cell wall integrity pathway (Gasch et al., 2000) and, unlike hydrogen peroxide, induces significant changes to the cell wall (Vilella et al., 2005). Thus, TUDCA can have differential effects on various chemicals and ER stressors. The absence of profound transcription changes in response to TUDCA argues that it must work rather quickly upon addition of Tm. Indeed pretreatment with TUDCA did not promote growth in Tm-containing plates (Figure 9A). Moreover, delayed addition of TUDCA following addition of Tm for 2 h did not restore growth (Figure 9B). Thus, in order to protect against Tm, TUDCA needs to be present together with the stressor.

TUDCA decreases drug bioavailability

Taken together, our data reveal two important aspects of TUDCA: 1) TUDCA can rescue cells from pharmacologic stressors and 2) to do so, TUDCA must be present at the time when the stressor is added. Our results suggest that TUDCA decreases intracellular drug bioavailability, probably by either modifying yeast cell wall integrity and/or directly interacting with drugs in the culture media. In support of the altered cell wall hypothesis, our transcriptome results identified cell wall components (TIR1, TIR3, AGA1, SAG1) among the differentially expressed genes, suggesting that TUDCA can affect cell wall composition. In support of the hypothesis that TUDCA might directly interact with Tm, other groups have reported that other bile acids can form micelles that can sequester amphiphilic drugs such as caspofungin and decrease their toxicity (Hsieh and Brock, 2017; Hsieh et al., 2017). Indeed, using a previously characterized assay for incorporation of coumarin-6 into micelles (Fluksman and Benny, 2019), we determined that the critical concentration for micelle formation of TUDCA is 5.17 mM (Figure 10A). Critically, only at micelle forming concentration were we able to observe TUDCA rescue of yeast growth in Tm (Figure 1A). In fact, and somewhat surprisingly, lower concentrations of TUDCA exacerbated the Tm-induced growth defect (Figure 10, B and C) suggesting TUDCA acts differently (probably as a cell wall stressor) on cells when used below the micelle forming concentration. TUDCA is present in vast excess over Tm, as 1 µg/ml Tm is 1.2 µM. As a result, we observed that the ability of TUDCA to alleviate Tm-induced UPR (measured with the UPR-mCherry fluorescent reporter) requires its addition at micelle forming concentration (Figure 10D). Collectively, our data support a model for Tm encapsulation into TUDCA micelles as a means to reduce its bioavailability, and consequently, its ability to induce ER stress.

It is difficult to directly measure the intracellular content of Tm. Fortuitously, we found in the course of our studies that TUDCA also interfered with the ability of common cellular dyes to stain cells. Taking advantage of this observation, we used dye labeling as a proxy to assess TUDCA's capacity to alter the bioavailability of compounds in general. We tested the efficacy of fluorescent dye labeling across a range of TUDCA concentrations. We found that simultaneous addition of TUDCA with either mitotracker (a mitochondrial staining dye) or FM4-64 dramatically decreased dye labeling, but only at 5 mM TUDCA, the minimal concentration that enables micelle formation (Supplemental Figure S4). TUDCA was present at concentrations several orders of magnitude higher than each dye (50 nM mitotracker and 8 µM FM4-64). Moreover, pretreatment with TUDCA followed by washout prior to staining was not sufficient to impair dye uptake (Supplemental Figure S5), suggesting TUDCA most directly impacts drug bioavailability. Together, our functional assays for Tm and visual assay for cell dyes suggest that micelle forming concentrations of TUDCA alleviates ER stress, by decreasing intracellular drug bioavailability, independently of any function of TUDCA as a chemical chaperone. Interestingly, both Tm and mitotracker are hydrophobic compounds solubilized in DMSO, while FM4-64 is hydrophilic. While it remains unclear what criteria define TUDCA susceptible compounds, hydrophobic compounds are likely to be targets, while hydrophilic compounds, such as DTT or FM4-64 are less predictable in terms of TUDCA susceptibility.

DISCUSSION

Unmitigated ER stress is deleterious across all organisms including yeast. The UPR monitors the fitness of the ER folding environment and adjusts components of the protein quality control machinery in response to changes in the misfolded protein burden. To regulate UPR activity, several different approaches are possible. First, UPR activity can be modulated by pharmacological UPR inhibitors or activators. Several small molecules that target ER stress sensors have been developed and are currently being tested in various ER stress–associated disease models (Gonzalez-Teuber et al., 2019). One could also potentially employ compounds or genetic approaches that act on either the UPR or other signaling pathways that function in parallel (Valenzuela et al., 2018). For example, activation of the heat shock response decreases UPR activity (Liu and Chang, 2008). Alternatively, it is possible to improve secretory protein folding using chemical chaperones (Perlmutter, 2002; Rajan et al., 2011). The question is which class (es) of regulators does TUDCA belong to?

The ER stressor Tm inhibits Alg7, an essential enzyme required for cell viability, which encodes the major target of Tm, the dolichyl-P–dependent N-acetylglucosamine-1-P transferase in the N-glycosylation pathway. Alg7 expression is specifically up-regulated in response to UPR activation (Barnes et al., 1984). Any compound that rescues cells from stress and impaired growth must somehow compensate for the loss of the dolichol pathway metabolites required for N-glycosylation of secretory proteins. N-glycosylation increases hydrophilicity of secretory proteins, can regulate steps in protein folding, and enables interactions with lectin partner proteins (Reily et al., 2019). Here, we show that TUDCA can decrease Tm-induced ER stress and improve cell growth. Surprisingly, TUDCA can do so in the absence of a functional UPR, suggesting a mechanism that circumvents the requirement for UPR signaling. Thus, TUDCA must either block Tm activity or overcome the action of Tm. This is an important finding, since few known mechanisms can overcome Tm. One such mechanism is the upregulation of ALG7 expression. In addition, deletion or depletion of the transporter responsible for Tm uptake into mammalian cells, MFSD2A, can also decrease Tm sensitivity (Bassik and Kampmann, 2011; Reiling et al., 2011). Whether a similar transporter exists in yeast is unclear. Our screen did not reveal an obvious candidate. Regardless, we find TUDCA quantitatively decreases the effects of Tm inhibition of N-linked glycosylation, at least at lower doses of Tm. Our data support a model of decreased availability/uptake of Tm (and other stressors and compounds). Because disruption of multidrug efflux transporters only modestly reduced TUDCA rescue, we hypothesize TUDCA likely decreases Tm internalization. Recent analysis of Tm-resistant aneuploid mutants revealed that the mutants have increased chitin content (Beaupere et al., 2018). We were surprised to observe only a few changes in the transcriptome of yeast treated with TUDCA during the 2 h time frame wherein we observe Tm resistance. If TUDCA causes significant cell wall stress, we would expect changes in the expression of CWI targets, which we did not observe. Similarly, the inability of TUDCA pretreatment to protect cells from Tm suggests that TUDCA must be present with the drug and that TUDCA's effect does not persist. These observations suggest TUDCA might affect cell wall/plasma membrane permeability via a nontranscriptional mechanism.

Interestingly, beyond the misfolded protein stress literature, there are reports of bile acids, including TUDCA, regulating drug absorption. Multiple mechanisms have been proposed to explain the ability of bile acids to modulate drug absorption, such as increasing drug solubility, regulation of drug transporters, and changing membrane permeability (Greer et al., 1998; Darkoh et al., 2010; Guan et al., 2011; Pavlović et al., 2018). While we observed up-regulation of multidrug transporters following TUDCA treatment, deletion of those same transporters had little impact on TUDCA rescue of Tm treatment (Supplemental Figure S2). In other eukaryotic cells, TUDCA molecules can localize to the plasma membrane at the lipid water interface (Mello-Vieira et al., 2013; Sheps et al., 2021). TUDCA treatment also affects fluidity and polarity of photoreceptor membranes (Sabat et al., 2021) and modulates membrane permeability in liposomes (Zhou et al., 2009).

Alternatively, TUDCA could directly interact with drugs/stressors themselves; bile acid salts can form complexes with drugs via ion-pairing or covalent conjugation through hydroxyl and carbonyl groups, resulting in increased drug solubility and availability (Pavlović et al., 2018). For example, conjugation of antibiotics such as kanamycin with bile acids results in increased toxicity for Staphylococcus aureus (Giovagnoli et al., 2017). Other bile acids can modulate antifungal drug toxicity by sequestering them into micelles (Hsieh et al., 2017). Prolonged treatment with other bile acids such as lithocholic acid can profoundly affect the yeast transcriptome, proteome and lipidome and increase longevity (Burstein et al., 2012; Beach et al., 2013, 2015). Long-term treatment with bile acids can also induce aneuploidy in yeast (Ferguson and Parry, 1984) which can lead to ER stress tolerance (Beaupere et al., 2018). Our data (especially Figure 10) support a model in which TUDCA micelles decrease bioavailability of Tm or other compounds. That is, at sufficiently high levels of TUDCA, cells experience decreased concentrations of some drugs, such that no stress response is required or enabling stress adaptation or hormesis to help cells survive normally lethal drug concentrations. It will be of interest for future studies to assess the prolonged effects of TUDCA treatment on the ER proteostasis network.

The path by which we have ultimately come to understand the mechanism of TUDCA highlights the value of a broad and systematic methodology for assessing future chemical chaperone candidates. Protection from stressors can involve mechanisms that have little to do with protein folding or stress pathways. We propose employing a combination of assays that measure not just cell stress reporters and growth assays, but directly measure protein damage, misfolding, and restoration of function across a range of stressor and putative chemical chaperone concentrations and timepoints. In several cases, protective effects only became apparent at longer timepoints (Figures 8 and 10) and lower doses of stressor (Figures 6 and 7). Similarly, other assays of cell stress or misfolded protein accumulation (Figures 2 and 3) gave distinct readouts of cell stress or actual levels of misfolded proteins. Any single assay could lead to a misleading conclusion. Our studies do not rule out potential cell or tissue protective roles for TUDCA in disease, but our studies provide little support for a chemical chaperone mode of action by TUDCA.

MATERIALS AND METHODS

Request a protocol through Bio-protocol

Drugs

H₂O₂ (426000010), DTT (R0861), MitotrackerRed (M46751), FM4-64 (F34653) were purchased from Thermo Fisher Scientific. TUDCA (580549), Tm (11089-65-9), and Diamide (10465-78-8) were from MilliporeSigma.

Strains, plasmids, and cell culture

Strains and plasmids used in this study are listed in Supplemental Tables S1 and S2. For every experiment, cells were thawed from frozen stocks and grown on YPD or selective SC agar plates at 30°C for 48 h before transferring to liquid culture. Cells were inoculated in 5 ml liquid media in polystyrene snap cap tubes, then grown overnight at 30°C in a rotating drum. OD₆₀₀ of cultured cells was measured using a spectrophotometer. Cells were diluted to a final concentration of OD₆₀₀ 0.2, then serially diluted five times. 5-fold dilutions were spotted onto agar plates and incubated at 30°C for 48 h before imaging.

Plasmids encoding CPY or CPY* (G255R) were constructed by cloning the CPY or CPY* sequences from CPY/CPY*-GFP plasmids (Promlek et al., 2011) into the SpeI/HindIII sites of P415-GAL1 (Mumberg et al., 1995). To generate fluorescently-tagged versions, yeast-optimized monomeric superfolder GFP (ymsfGFP) (Jiang et al., 2017) was inserted downstream of the CPY/CPY* coding sequence using the HindIII/SalI sites of P415-GAL1. Primers are listed in Supplemental Table S3.

Liquid growth assay

Cells were inoculated in 5 ml liquid media in polystyrene snap cap tubes, then grown overnight at 30°C in a rotating drum. OD₆₀₀ of cultured cells was measured. Cells were diluted to a final concentration of OD₆₀₀ 0.15 in a flat-bottom 96-well plate and loaded into a BioTek plate reader in triplicate. Plates were held at 30°C with constant shaking and OD₆₀₀ measurements were taken every 15 min.

Flow cytometry

Flow cytometry was used to measure fluorescence from fluorescent reporter-expressing strains as well as to measure fluorescent dye internalization. Experiments were performed using a BD FACS Celesta equipped with the FACS Diva software. Strains were grown in 50 ml flasks to mid-log phase (OD₆₀₀ ∼0.5), then treated as indicated (Tm, TUDCA, or both) for indicated time periods before measurement of fluorescence using a PE-TexasRed filter. For time course experiments, samples were loaded in triplicate into 96-well plates and measured using the BD FACS Celesta HTS plate reader system; 20 µl samples were taken at each time point and fluorescence data for a maximum of 10,000 cells were recorded. Between measurements, plates were incubated at 30°C with constant shaking. Median fluorescence data for each sample were used for analysis. No gates were applied.

Protein isolation and immunoblot

Whole-cell protein was isolated using alkaline lysis (Kushnirov, 2000). Mid-log phase cells were treated as indicated, OD₆₀₀ was measured, and an aliquot of cells equivalent of OD₆₀₀ = 1.0 was taken from each sample. Cells were pelleted then resuspended in 200 µl 0.1 M NaOH and incubated at room temperature for 5 min. Cells were pelleted again, then resuspended in 50 µl of sample buffer (4x Laemmeli buffer, water, and 1 M DTT) before boiling at 100°C for 3 min. Samples were pelleted and the supernatant was transferred to new 1.5 ml tubes.

Proteins were loaded into BioRad TGX Stain-Free gels and separated by gel electrophoresis. Stain-free technology was used as a loading control by activating the gel with UV light for 5 min before transfer. Proteins were transferred to nitrocellulose membranes using a BioRad Turbo Blot system and imaged with UV light to obtain final stain-free images. Membranes were incubated at 4°C overnight with anti-Pdi1 antibody (Santa Cruz Biotechnology (sc-57963), 1/5000 in 5% powdered skim milk) then probed with secondary antibodies (LI-COR goat anti-mouse) for 1 h at room temperature before imaging. Densitometry analysis was conducted using Image Lab software.

Northern blot

RNA levels were assessed using Northern blot as described previously (Lajoie et al., 2012). Briefly, early-log cells were treated with Tm and TUDCA and cells were pelleted and frozen in dry ice. Total RNA was isolated using the hot phenol method. RNA was run on formaldehyde gels and transferred to Nytran Plus membrane. Blots were probed with ³²P-labeled oligonucleotide and imaged using 820 Phosphorimager (GE Healthcare).

Deletion library screens

Using the ROTOR HDA robotic system (Singer Instruments), frozen stock yeast deletion libraries were thawed and spotted from liquid cultures onto agar plates using 96- or 384-pin pads. Plates were incubated at 30°C for 1–2 d, then repinned onto agar plates containing indicated treatments and incubated at 30°C once again. Plates were imaged using a Nikon camera dock. Colony size was quantified using ColonyImager (S&P Robotics), and ratios of growth rescue were calculated by dividing relative colony size on Tm + TUDCA plates by colony size on plates containing TUDCA alone. Deletions that showed 50% growth reduction on Tm in presence of TUDCA compared with wild-type were retested in duplicate to confirm the phenotype.

RNA sequencing and transcriptome analysis

RNA was isolated from two biological replicates from early log phase growth in either YPD or YPD containing 5 mM TUDCA using the MasterPure Yeast RNA purification kit (Lucigen) according to the manufacturer's instructions. Total RNA sequencing was performed by Genewiz (South Plainfield, NJ). Stranded Illumina TruSeq cDNA libraries with poly dT enrichment were prepared from high-quality total RNA (RIN > 8). Libraries were sequenced on an Illumina HiSeq, yielding between 100 and 124 million 150 bp paired end sequencing reads per sample. The raw data have been deposited in NCBI's Gene Expression Omnibus (GEO; Edgar et al., 2002) and are accessible through GEO Series accession number GSE186390.

FASTQ files were analyzed with a customized bioinformatics workflow. Adapter sequences were trimmed using Trimmomatic (Bolger et al., 2014) and aligned to the S. cerevisiae S288C reference genome (assembly R64-2-1; https://www.yeastgenome.org/) using STAR (Dobin et al., 2013). Reads that were uniquely mapped to the reference genome were counted for each gene using featureCounts (Liao et al., 2014). Differential expression analysis was performed using the DESeq2 R package (Love et al., 2014) with a Benjamini–Hochberg adjusted p-value cutoff of ≤ 0.05. GO analysis was performed using the Saccharomyces Genome Database (SGD) (Cherry et al., 2012) GO Term Finder using p < 0.01.

Micelle formation assay

Critical micelle concentration was measured using coumarin-6 (Fluksman and Benny, 2019). A 6 µM solution of coumarin-6 was prepared in dichloromethane and 40 µl of the stock solution was distributed to Eppendorf tubes and allowed to evaporate in a fume hood for 30 min. TUDCA solutions were prepared in dH₂O ranging from 0 to 10 mM concentrations. Each TUDCA solution was added to a tube containing dried coumarin-6 and rotated in the dark at room temperature for 20 h. After the incubation period, 200 µl of each TUDCA-coumarin-6 solution was transferred to the wells of a 96-well plate and fluorescence intensity was read using excitation and emission wavelength filters of 485 ± 20 nm and 528 ± 20 nm, respectively, on a Cytation5 (BioTek). The resulting fluorescence intensities were plotted against the corresponding TUDCA concentration in GraphPad Prism. The critical micellar concentration was determined by identifying the point of intersection of the two tangents created by the graph. Points on the graph are representative of the average of three replicates.

FRAP

Live Kar2-sfGFP cells (Lajoie et al., 2012) were imaged on a Duoscan confocal microscope system (Carl Zeiss Microimaging) equipped with a 63 ×, numerical aperture 1.4 oil objective and a 489 nm, 100 mW diode laser with a 500 to 550 nm bandpass filter for GFP. FRAP was performed by photobleaching a region of interest at full laser power of the 489 nm line and monitoring fluorescence recovery over time. Diffusion coefficient (D) values were determined using an inhomogeneous diffusion simulation, as described previously (Siggia et al., 2000; Snapp and Lajoie, 2011).

Fluorescence microscopy

Cells were inoculated in 3 ml of liquid media and grown overnight at 30°C in a shaking incubator. A total of 200 µl aliquots were separated into 1.5 ml tubes and treated with TUDCA for 15 min. Fluorescent dyes FM4-64, and Mitrotracker were added to the cell suspension and imaged. A total of 2 µl of stained cells were added to a glass slide beneath a glass coverslip. Live cell fluorescence imaging was conducted on the Cytation5 (BioTek). FM4-64, Mitotracker and azole-conjugated dyes stained cells were imaged using the TexasRed filter using a 20 × Plan Extended Apochromat. NA 0.8.

CPY/CPY*-ymsfGFP was imaged using a Zeiss Axiovert A1 wide-field fluorescence microscope with 63 × 1.4 NA oil objective using a GFP filter (excitation 470/40, emission 525/50) and an AxioCam ICm1 R1 CCD camera. ImageJ was used to analyze the images (Schneider et al., 2012).

FOOTNOTES

REFERENCES

• Albakri MB, Jiang Y, Genereaux J, Lajoie P (2018). Polyglutamine toxicity assays highlight the advantages of mScarlet for imaging in Saccharomyces cerevisiae. F1000Res 7, 1242. Crossref, Medline, Google Scholar
• Babour A, Bicknell AA, Tourtellotte J, Niwa M (2010). A surveillance pathway monitors the fitness of the endoplasmic reticulum to control its inheritance. Cell 142, 256–269. Crossref, Medline, Google Scholar
• Balch WE, Morimoto RI, Dillin A, Kelly JW (2008). Adapting proteostasis for disease intervention. Science 319, 916–919. Crossref, Medline, Google Scholar
• Barnes G, Hansen WJ, Holcomb CL, Rine J (1984). Asparagine-linked glycosylation in Saccharomyces cerevisiae: Genetic analysis of an early step. Mol Cell Biol 4, 2381–2388. Crossref, Medline, Google Scholar
• Bassik MC, Kampmann M (2011). Knocking out the door to tunicamycin entry. Proc Natl Acad Sci U S A 108, 11731–11732. Crossref, Medline, Google Scholar
• Beach A, Richard VR, Bourque S, Boukh-Viner T, Kyryakov P, Gomez-Perez A, Arlia-Ciommo A, Feldman R, Leonov A, Piano A, et al. (2015). Lithocholic bile acid accumulated in yeast mitochondria orchestrates a development of an anti-aging cellular pattern by causing age-related changes in cellular proteome. Cell Cycle 14, 1643–1656. Crossref, Medline, Google Scholar
• Beach A, Richard VR, Leonov A, Burstein MT, Bourque SD, Koupaki O, Juneau M, Feldman R, Iouk T, Titorenko VI (2013). Mitochondrial membrane lipidome defines yeast longevity. Aging 5, 551–574. Crossref, Medline, Google Scholar
• Beaupere C, Dinatto L, Wasko BM, Chen RB, VanValkenburg L, Kiflezghi MG, Lee MB, Promislow DEL, Dang W, Kaeberlein M, et al. (2018). Genetic screen identifies adaptive aneuploidy as a key mediator of ER stress resistance in yeast. Proc Natl Acad Sci U S A 115, 9586–9591. Crossref, Medline, Google Scholar
• Berger E, Haller D (2011). Structure-function analysis of the tertiary bile acid TUDCA for the resolution of endoplasmic reticulum stress in intestinal epithelial cells. Biochem Biophys Res Commun 409, 610–615. Crossref, Medline, Google Scholar
• Boatright JH, Nickerson JM, Moring AG, Pardue MT (2009). Bile acids in treatment of ocular disease. J Ocul Biol Dis Infor 2, 149–159. Crossref, Medline, Google Scholar
• Bolger AM, Lohse M, Usadel B (2014). Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120. Crossref, Medline, Google Scholar
• Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P, Boeke JD (1998). Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132. Crossref, Medline, Google Scholar
• Burstein MT, Kyryakov P, Beach A, Richard VR, Koupaki O, Gomez-Perez A, Leonov A, Levy S, Noohi F, Titorenko VI (2012). Lithocholic acid extends longevity of chronologically aging yeast only if added at certain critical periods of their lifespan. Cell Cycle 11, 3443–3462. Crossref, Medline, Google Scholar
• Cao SS, Zimmermann EM, Chuang B-M, Song B, Nwokoye A, Wilkinson JE, Eaton KA, Kaufman RJ (2013). The unfolded protein response and chemical chaperones reduce protein misfolding and colitis in mice. Gastroenterology 144, 989–1000.e6. Crossref, Medline, Google Scholar
• Celik C, Lee SYT, Yap WS, Thibault G (2023). Endoplasmic reticulum stress and lipids in health and diseases. Prog Lipid Res 89, 101198. Crossref, Medline, Google Scholar
• Chadwick SR, Lajoie P (2019). Endoplasmic reticulum stress coping mechanisms and lifespan regulation in health and diseases. Front Cell Dev Biol 7, 84. Crossref, Medline, Google Scholar
• Chawla A, Chakrabarti S, Ghosh G, Niwa M (2011). Attenuation of yeast UPR is essential for survival and is mediated by IRE1 kinase. J Cell Biol 193, 41–50. Crossref, Medline, Google Scholar
• Cherry JM, Hong EL, Amundsen C, Balakrishnan R, Binkley G, Chan ET, Christie KR, Costanzo MC, Dwight SS, Engel SR, et al. (2012). Saccharomyces Genome Database: The genomics resource of budding yeast. Nucleic Acids Res 40, D700–D705. Crossref, Medline, Google Scholar
• Costanzo M, VanderSluis B, Koch EN, Baryshnikova A, Pons C, Tan G, Wang W, Usaj M, Hanchard J, Lee SD, et al. (2016). A global genetic interaction network maps a wiring diagram of cellular function. Science 353, aaf1420. Crossref, Medline, Google Scholar
• Cox JS, Shamu CE, Walter P (1993). Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell 73, 1197–1206. Crossref, Medline, Google Scholar
• Cox JS, Walter P (1996). A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell 87, 391–404. Crossref, Medline, Google Scholar
• Cremers CM, Knoefler D, Vitvitsky V, Banerjee R, Jakob U (2014). Bile salts act as effective protein-unfolding agents and instigators of disulfide stress in vivo. Proc Natl Acad Sci U S A 111, E1610–E1619. Crossref, Medline, Google Scholar
• Cronin SR, Rao R, Hampton RY (2002). Cod1p/Spf1p is a P-type ATPase involved in ER function and Ca2+ homeostasis. J Cell Biol 157, 1017–1028. Crossref, Medline, Google Scholar
• Darkoh C, Lichtenberger LM, Ajami N, Dial EJ, Jiang Z-D, DuPont HL (2010). Bile acids improve the antimicrobial effect of rifaximin. Antimicrob Agents Chemother 54, 3618–3624. Crossref, Medline, Google Scholar
• Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013). STAR: Ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21. Crossref, Medline, Google Scholar
• Drickamer K (1988). Two distinct classes of carbohydrate-recognition domains in animal lectins. J Biol Chem 263, 9557–9560. Crossref, Medline, Google Scholar
• Edgar R, Domrachev M, Lash AE (2002). Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 30, 207–210. Crossref, Medline, Google Scholar
• Einstein A (1905). Über die von der molekularkinetischen Theorie der Wärme geforderte Bewegung von in ruhenden Flüssigkeiten suspendierten Teilchen. Ann Phys 322, 549–560. Crossref, Google Scholar
• Ferguson LR, Parry JM (1984). Mitotic aneuploidy as a possible mechanism for tumour promoting activity in bile acids. Carcinogenesis 5, 447–451. Crossref, Medline, Google Scholar
• Finger A, Knop M, Wolf DH (1993). Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast. Eur J Biochem 218, 565–574. Crossref, Medline, Google Scholar
• Fluksman A, Benny O (2019). A robust method for critical micelle concentration determination using coumarin-6 as a fluorescent probe. Anal Methods 11, 3810–3818. Crossref, Google Scholar
• Fun XH, Thibault G (2020). Lipid bilayer stress and proteotoxic stress-induced unfolded protein response deploy divergent transcriptional and non-transcriptional programmes. Biochim Biophys Acta Mol Cell Biol Lipids 1865, 158449. Crossref, Medline, Google Scholar
• Gallagher CM, Walter P (2016). Ceapins inhibit ATF6α signaling by selectively preventing transport of ATF6α to the Golgi apparatus during ER stress. Elife 5, e11880. Crossref, Medline, Google Scholar
• García-González P, Cabral-Miranda F, Hetz C, Osorio F (2018). Interplay between the unfolded protein response and immune function in the development of neurodegenerative diseases. Front Immunol 9, 2541. Crossref, Medline, Google Scholar
• Gardner BM, Walter P (2011). Unfolded proteins are Ire1-activating ligands that directly induce the unfolded protein response. Science 333, 1891–1894. Crossref, Medline, Google Scholar
• Gasch AP, Spellman PT, Kao CM, Carmel-Harel O, Eisen MB, Storz G, Botstein D, Brown PO (2000). Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell 11, 4241–4257. Link, Google Scholar
• Giaever G, Chu AM, Ni L, Connelly C, Riles L, Véronneau S, Dow S, Lucau-Danila A, Anderson K, André B, et al. (2002). Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391. Crossref, Medline, Google Scholar
• Giovagnoli S, Pietrella D, Barberini L, Santi C, Carotti A, di Michele A, Ricci M (2017). Reshaping antibiotics through hydrophobic drug-bile acid ionic complexation enhances activity against Staphylococcus aureus biofilms. Int J Pharm 528, 144–162. Crossref, Medline, Google Scholar
• Gonzalez-Teuber V, Albert-Gasco H, Auyeung VC, Papa FR, Mallucci GR, Hetz C (2019). Small molecules to improve ER proteostasis in disease. Trends Pharmacol Sci 40, 684–695. Crossref, Medline, Google Scholar
• Grandjean JMD, Wiseman RL (2020). Small molecule strategies to harness the unfolded protein response: Where do we go from here? J Biol Chem 295, 15692–15711. Crossref, Medline, Google Scholar
• Greer FR, Marshall SP, Severson RR, Smith DA, Shearer MJ, Pace DG, Joubert PH (1998). A new mixed micellar preparation for oral vitamin K prophylaxis: Randomised controlled comparison with an intramuscular formulation in breast fed infants. Arch Dis Child 79, 300–305. Crossref, Medline, Google Scholar
• Guan P, Lu Y, Qi J, Niu M, Lian R, Hu F, Wu W (2011). Enhanced oral bioavailability of cyclosporine A by liposomes containing a bile salt. Int J Nanomedicine 6, 965–974. Medline, Google Scholar
• Halbleib K, Pesek K, Covino R, Hofbauer HF, Wunnicke D, Hänelt I, Hummer G, Ernst R (2017). Activation of the unfolded protein response by lipid bilayer stress. Mol Cell 67, 673–684.e8. Crossref, Medline, Google Scholar
• Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, Ron D (2000). Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell 6, 1099–1108. Crossref, Medline, Google Scholar
• Hetz C, Axten JM, Patterson JB (2019a). Pharmacological targeting of the unfolded protein response for disease intervention. Nat Chem Biol 15, 764–775. Crossref, Medline, Google Scholar
• Hetz C, Axten JM, Patterson JB (2019b). Publisher Correction: Pharmacological targeting of the unfolded protein response for disease intervention. Nat Chem Biol 15, 1129. Crossref, Medline, Google Scholar
• Hetz C, Papa FR (2018). The unfolded protein response and cell fate control. Mol Cell 69, 169–181. Crossref, Medline, Google Scholar
• Ho N, Yap WS, Xu J, Wu H, Koh JH, Goh WWB, George B, Chong SC, Taubert S, Thibault G (2020). Stress sensor Ire1 deploys a divergent transcriptional program in response to lipid bilayer stress. J Cell Biol 219, e201909165. Crossref, Medline, Google Scholar
• Hou Y, Luan J, Huang T, Deng T, Li X, Xiao Z, Zhan J, Luo D, Hou Y, Xu L, et al. (2021). Tauroursodeoxycholic acid alleviates secondary injury in spinal cord injury mice by reducing oxidative stress, apoptosis, and inflammatory response. J Neuroinflammation 18, 216. Crossref, Medline, Google Scholar
• Hsieh S-H, Brock M (2017). Lipid components of bile increase the protective effect of conjugated bile salts against antifungal drugs. Fungal Biol 121, 929–938. Crossref, Medline, Google Scholar
• Hsieh S-H, Brunke S, Brock M (2017). Encapsulation of antifungals in micelles protects Candida albicans during gall-bladder infection. Front Microbiol 8, 117. Crossref, Medline, Google Scholar
• Igbaria A, Merksamer PI, Trusina A, Tilahun F, Johnson JR, Brandman O, Krogan NJ, Weissman JS, Papa FR (2019). Chaperone-mediated reflux of secretory proteins to the cytosol during endoplasmic reticulum stress. Proc Natl Acad Sci U S A 116, 11291–11298. Crossref, Medline, Google Scholar
• Ishiwata-Kimata Y, Le QG, Kimata Y (2021). Induction and aggravation of the endoplasmic-reticulum stress by membrane-lipid metabolic intermediate phosphatidyl–monomethylethanolamine. Front Cell Dev Biol 9, 743018. Crossref, Medline, Google Scholar
• Jiang Y, Chadwick SR, Lajoie P (2016). Endoplasmic reticulum stress: The cause and solution to Huntington's disease? Brain Res 1648, 650–657. Crossref, Medline, Google Scholar
• Jiang Y, Di Gregorio SE, Duennwald ML, Lajoie P (2017). Polyglutamine toxicity in yeast uncovers phenotypic variations between different fluorescent protein fusions. Traffic 18, 58–70. Crossref, Medline, Google Scholar
• Jonikas MC, Collins SR, Denic V, Oh E, Quan EM, Schmid V, Weibezahn J, Schwappach B, Walter P, Weissman JS, et al. (2009). Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum. Science 323, 1693–1697. Crossref, Medline, Google Scholar
• Karagöz GE, Acosta-Alvear D, Nguyen HT, Lee CP, Chu F, Walter P (2017). An unfolded protein-induced conformational switch activates mammalian IRE1. Elife 6, e30700. Crossref, Medline, Google Scholar
• Keene CD, Rodrigues CMP, Eich T, Chhabra MS, Steer CJ, Low WC (2002). Tauroursodeoxycholic acid, a bile acid, is neuroprotective in a transgenic animal model of Huntington's disease. Proc Natl Acad Sci U S A 99, 10671–10676. Crossref, Medline, Google Scholar
• Kelly JW (2020). Pharmacologic approaches for adapting proteostasis in the secretory pathway to ameliorate protein conformational diseases. Cold Spring Harb Perspect Biol 12, a034108. Crossref, Medline, Google Scholar
• Kuo S-C, Lampen JO (1974). Tunicamycin — An inhibitor of yeast glycoprotein synthesis. Biochem Biophys Res Commun 58, 287–295. Crossref, Medline, Google Scholar
• Kusaczuk M (2019). Tauroursodeoxycholate-bile acid with chaperoning activity: Molecular and cellular effects and therapeutic perspectives. Cells 8, 1471. Crossref, Medline, Google Scholar
• Kushnirov VV (2000). Rapid and reliable protein extraction from yeast. Yeast 16, 857–860. Crossref, Medline, Google Scholar
• Lai CW, Aronson DE, Snapp EL (2010). BiP availability distinguishes states of homeostasis and stress in the endoplasmic reticulum of living cells. Mol Biol Cell 21, 1909–1921. Link, Google Scholar
• Lajoie P, Moir RD, Willis IM, Snapp EL (2012). Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells. Mol Biol Cell 23, 955–964. Link, Google Scholar
• Lajoie P, Snapp EL (2011). Changes in BiP availability reveal hypersensitivity to acute endoplasmic reticulum stress in cells expressing mutant huntingtin. J Cell Sci 124, 3332–3343. Crossref, Medline, Google Scholar
• Lajoie P, Snapp EL (2020). Size-dependent secretory protein reflux into the cytosol in association with acute endoplasmic reticulum stress. Traffic 21, 419–429. Crossref, Medline, Google Scholar
• Lawson EC, Bhatia SK, Han MK, Aung MH, Ciavatta V, Boatright JH, Pardue MT (2016). Tauroursodeoxycholic acid protects retinal function and structure in rd1 mice. Adv Exp Med Biol 854, 431–436. Crossref, Medline, Google Scholar
• Liao Y, Smyth GK, Shi W (2014). featureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930. Crossref, Medline, Google Scholar
• Li S, Tan HY, Wang N, Hong M, Li L, Cheung F, Feng Y (2016). Substitutes for bear bile for the treatment of liver diseases: Research progress and future perspective. Evid Based Complement Alternat Med 2016, 4305074. Crossref, Medline, Google Scholar
• Liu Y, Chang A (2008). Heat shock response relieves ER stress. EMBO J 27, 1049–1059. Crossref, Medline, Google Scholar
• Lojpur T, Easton Z, Raez-Villanueva S, Laviolette S, Holloway AC, Hardy DB (2019). Δ9-Tetrahydrocannabinol leads to endoplasmic reticulum stress and mitochondrial dysfunction in human BeWo trophoblasts. Reprod Toxicol 87, 21–31. Crossref, Medline, Google Scholar
• Lopez AD, Tar K, Krügel U, Dange T, Ros IG, Schmidt M (2011). Proteasomal degradation of Sfp1 contributes to the repression of ribosome biogenesis during starvation and is mediated by the proteasome activator Blm10. Mol Biol Cell 22, 528–540. Link, Google Scholar
• Love MI, Huber W, Anders S (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550. Crossref, Medline, Google Scholar
• Mai CT, Le QG, Ishiwata-Kimata Y, Takagi H, Kohno K, Kimata Y (2018). 4-Phenylbutyrate suppresses the unfolded protein response without restoring protein folding in Saccharomyces cerevisiae. FEMS Yeast Res 18. Crossref, Medline, Google Scholar
• Mai TC, Ishiwata-Kimata Y, Le QG, Kido H, Kimata Y (2019). Dispersion of endoplasmic reticulum-associated compartments by 4-phenyl butyric acid in yeast cells. Cell Struct Funct 44, 173–182. Crossref, Medline, Google Scholar
• Maly DJ, Papa FR (2014). Druggable sensors of the unfolded protein response. Nat Chem Biol 10, 892–901. Crossref, Medline, Google Scholar
• Marciniak SJ, Chambers JE, Ron D (2022). Pharmacological targeting of endoplasmic reticulum stress in disease. Nat Rev Drug Discov 21, 115–140. Crossref, Medline, Google Scholar
• Marion RM, Regev A, Segal E, Barash Y, Koller D, Friedman N, O'Shea EK (2004). Sfp1 is a stress- and nutrient-sensitive regulator of ribosomal protein gene expression. Proc Natl Acad Sci U S A 101, 14315–14322. Crossref, Medline, Google Scholar
• Ma W, Goldberg E, Goldberg J (2017). ER retention is imposed by COPII protein sorting and attenuated by 4-phenylbutyrate. Elife 6, e26624. Crossref, Medline, Google Scholar
• Mello-Vieira J, Sousa T, Coutinho A, Fedorov A, Lucas SD, Moreira R, Castro RE, Rodrigues CMP, Prieto M, Fernandes F (2013). Cytotoxic bile acids, but not cytoprotective species, inhibit the ordering effect of cholesterol in model membranes at physiologically active concentrations. Biochim Biophys Acta 1828, 2152–2163. Crossref, Medline, Google Scholar
• Merksamer PI, Trusina A, Papa FR (2008). Real-time redox measurements during endoplasmic reticulum stress reveal interlinked protein folding functions. Cell 135, 933–947. Crossref, Medline, Google Scholar
• Mumberg D, Müller R, Funk M (1995). Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156, 119–122. Crossref, Medline, Google Scholar
• Nunes AF, Amaral JD, Lo AC, Fonseca MB, Viana RJS, Callaerts-Vegh Z, D'Hooge R, Rodrigues CMP (2012). TUDCA, a bile acid, attenuates amyloid precursor protein processing and amyloid-β deposition in APP/PS1 mice. Mol Neurobiol 45, 440–454. Crossref, Medline, Google Scholar
• Omura T, Asari M, Yamamoto J, Oka K, Hoshina C, Maseda C, Awaya T, Tasaki Y, Shiono H, Yonezawa A, et al. (2013). Sodium tauroursodeoxycholate prevents paraquat-induced cell death by suppressing endoplasmic reticulum stress responses in human lung epithelial A549 cells. Biochem Biophys Res Commun 432, 689–694. Crossref, Medline, Google Scholar
• Ozcan U, Yilmaz E, Ozcan L, Furuhashi M, Vaillancourt E, Smith RO, Görgün CZ, Hotamisligil GS (2006). Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes. Science 313, 1137–1140. Crossref, Medline, Google Scholar
• Papp E, Csermely P (2006). Chemical chaperones: Mechanisms of action and potential use. Handb Exp Pharmacol 172, 405–416. Medline, Google Scholar
• Pavlović N, Goločorbin-Kon S, Ðanić M, Stanimirov B, Al-Salami H, Stankov K, Mikov M (2018). Bile acids and their derivatives as potential modifiers of drug release and pharmacokinetic profiles. Front Pharmacol 9, 1283. Crossref, Medline, Google Scholar
• Perlmutter DH (2002). Chemical chaperones: A pharmacological strategy for disorders of protein folding and trafficking. Pediatr Res 52, 832–836. Crossref, Medline, Google Scholar
• Pincus D, Aranda-Díaz A, Zuleta IA, Walter P, El-Samad H (2014). Delayed Ras/PKA signaling augments the unfolded protein response. Proc Natl Acad Sci U S A 111, 14800–14805. Crossref, Medline, Google Scholar
• Pioltine EM, Costa CB, Barbosa Latorraca L, Franchi FF, Dos Santos PH, Mingoti GZ, de Paula-Lopes FF, Nogueira MFG (2021). Treatment of in vitro-matured bovine oocytes with tauroursodeoxycholic acid modulates the oxidative stress signaling pathway. Front Cell Dev Biol 9, 623852. Crossref, Medline, Google Scholar
• Promlek T, Ishiwata-Kimata Y, Shido M, Sakuramoto M, Kohno K, Kimata Y (2011). Membrane aberrancy and unfolded proteins activate the endoplasmic reticulum stress sensor Ire1 in different ways. Mol Biol Cell 22, 3520–3532. Link, Google Scholar
• Qiao X, Ye M, Pan D-L, Miao W-J, Xiang C, Han J, Guo D-A (2011). Differentiation of various traditional Chinese medicines derived from animal bile and gallstone: Simultaneous determination of bile acids by liquid chromatography coupled with triple quadrupole mass spectrometry. J Chromatogr A 1218, 107–117. Crossref, Medline, Google Scholar
• Ragni E, Piberger H, Neupert C, García-Cantalejo J, Popolo L, Arroyo J, Aebi M, Strahl S (2011). The genetic interaction network of CCW12, a Saccharomyces cerevisiae gene required for cell wall integrity during budding and formation of mating projections. BMC Genomics 12, 107. Crossref, Medline, Google Scholar
• Rai S, Szaruga M, Pitera AP, Bertolotti A (2024). Integrated stress response activator halofuginone protects mice from diabetes-like phenotypes. J Cell Biol 223, e202405175. Crossref, Medline, Google Scholar
• Rajan RS, Tsumoto K, Tokunaga M, Tokunaga H, Kita Y, Arakawa T (2011). Chemical and pharmacological chaperones: Application for recombinant protein production and protein folding diseases. Curr Med Chem 18, 1–15. Crossref, Medline, Google Scholar
• Reiling JH, Clish CB, Carette JE, Varadarajan M, Brummelkamp TR, Sabatini DM (2011). A haploid genetic screen identifies the major facilitator domain containing 2A (MFSD2A) transporter as a key mediator in the response to tunicamycin. Proc Natl Acad Sci U S A 108, 11756–11765. Crossref, Medline, Google Scholar
• Reily C, Stewart TJ, Renfrow MB, Novak J (2019). Glycosylation in health and disease. Nat Rev Nephrol 15, 346–366. Crossref, Medline, Google Scholar
• Ren J, Bi Y, Sowers JR, Hetz C, Zhang Y (2021). Endoplasmic reticulum stress and unfolded protein response in cardiovascular diseases. Nat Rev Cardiol 18, 499–521. Crossref, Medline, Google Scholar
• Rivard AL, Steer CJ, Kren BT, Rodrigues CMP, Castro RE, Bianco RW, Low WC (2007). Administration of tauroursodeoxycholic acid (TUDCA) reduces apoptosis following myocardial infarction in rat. Am J Chin Med 35, 279–295. Crossref, Medline, Google Scholar
• Rogers B, Decottignies A, Kolaczkowski M, Carvajal E, Balzi E, Goffeau A (2001). The pleitropic drug ABC transporters from Saccharomyces cerevisiae. J Mol Microbiol Biotechnol 3, 207–214. Medline, Google Scholar
• Rose MD, Misra LM, Vogel JP (1989). KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene. Cell 57, 1211–1221. Crossref, Medline, Google Scholar
• Rubio C, Pincus D, Korennykh A, Schuck S, El-Samad H, Walter P (2011). Homeostatic adaptation to endoplasmic reticulum stress depends on Ire1 kinase activity. J Cell Biol 193, 171–184. Crossref, Medline, Google Scholar
• Rutkowski DT, Arnold SM, Miller CN, Wu J, Li J, Gunnison KM, Mori K, Sadighi Akha AA, Raden D, Kaufman RJ (2006). Adaptation to ER stress is mediated by differential stabilities of pro-survival and pro-apoptotic mRNAs and proteins. PLoS Biol 4, e374. Crossref, Medline, Google Scholar
• Sabat MJ, Wiśniewska-Becker AM, Markiewicz M, Marzec KM, Dybas J, Furso J, Pabisz P, Duda M, Pawlak AM (2021). Tauroursodeoxycholic acid (TUDCA)—lipid interactions and antioxidant properties of TUDCA studied in model of photoreceptor membranes. Membranes 11, 327. Crossref, Medline, Google Scholar
• Schmidt RM, Schessner JP, Borner GH, Schuck S (2019). The proteasome biogenesis regulator Rpn4 cooperates with the unfolded protein response to promote ER stress resistance. Elife 8, e43244. Crossref, Medline, Google Scholar
• Schneider CA, Rasband WS, Eliceiri KW (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9, 671–675. Crossref, Medline, Google Scholar
• Shamu CE, Walter P (1996). Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus. EMBO J 15, 3028–3039. Crossref, Medline, Google Scholar
• Sheps JA, Wang R, Wang J, Ling V (2021). The protective role of hydrophilic tetrahydroxylated bile acids (THBA). Biochim Biophys Acta Mol Cell Biol Lipids 1866, 158925. Crossref, Medline, Google Scholar
• Siggia ED, Lippincott-Schwartz J, Bekiranov S (2000). Diffusion in inhomogeneous media: theory and simulations applied to whole cell photobleach recovery. Biophys J 79, 1761–1770. Crossref, Medline, Google Scholar
• Silberstein S, Schlenstedt G, Silver PA, Gilmore R (1998). A role for the DnaJ homologue Scj1p in protein folding in the yeast endoplasmic reticulum. J Cell Biol 143, 921–933. Crossref, Medline, Google Scholar
• Singh LR, Chen X, Kozich V, Kruger WD (2007). Chemical chaperone rescue of mutant human cystathionine beta-synthase. Mol Genet Metab 91, 335–342. Crossref, Medline, Google Scholar
• Snapp EL, Lajoie P (2011). Photobleaching regions of living cells to monitor membrane traffic. Cold Spring Harb Protoc 2011, 1366–1367. Medline, Google Scholar
• Spear ED, Ng DTW (2003). Stress tolerance of misfolded carboxypeptidase Y requires maintenance of protein trafficking and degradative pathways. Mol Biol Cell 14, 2756–2767. Link, Google Scholar
• Tachikawa H, Miura T, Katakura Y, Mizunaga T (1991). Molecular structure of a yeast gene, PDI1, encoding protein disulfide isomerase that is essential for cell growth. J Biochem 110, 306–313. Crossref, Medline, Google Scholar
• Tatu U, Braakman I, Helenius A (1993). Membrane glycoprotein folding, oligomerization and intracellular transport: Effects of dithiothreitol in living cells. EMBO J 12, 2151–2157. Crossref, Medline, Google Scholar
• Taylor RC, Hetz C (2020). Mastering organismal aging through the endoplasmic reticulum proteostasis network. Aging Cell 19, e13265. Crossref, Medline, Google Scholar
• Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JS, Walter P (2000). Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101, 249–258. Crossref, Medline, Google Scholar
• Uppala JK, Gani AR, Ramaiah KVA (2017). Chemical chaperone, TUDCA unlike PBA, mitigates protein aggregation efficiently and resists ER and non-ER stress induced HepG2 cell death. Sci Rep 7, 3831. Crossref, Medline, Google Scholar
• Usaj M, Tan Y, Wang W, VanderSluis B, Zou A, Myers CL, Costanzo M, Andrews B, Boone C (2017). TheCellMap.org: A web-accessible database for visualizing and mining the global yeast genetic interaction network. G3 7, 1539–1549. Crossref, Medline, Google Scholar
• Valenzuela V, Jackson KL, Sardi SP, Hetz C (2018). Gene therapy strategies to restore ER proteostasis in disease. Mol Ther 26, 1404–1413. Crossref, Medline, Google Scholar
• Varki A (2017). Biological roles of glycans. Glycobiology 27, 3–49. Crossref, Medline, Google Scholar
• Vilella F, Herrero E, Torres J, de la Torre-Ruiz MA (2005). Pkc1 and the upstream elements of the cell integrity pathway in Saccharomyces cerevisiae, Rom2 and Mtl1, are required for cellular responses to oxidative stress. J Biol Chem 280, 9149–9159. Crossref, Medline, Google Scholar
• Volmer R, van der Ploeg K, Ron D (2013). Membrane lipid saturation activates endoplasmic reticulum unfolded protein response transducers through their transmembrane domains. Proc Natl Acad Sci U S A 110, 4628–4633. Crossref, Medline, Google Scholar
• Volmer R, Ron D (2015). Lipid-dependent regulation of the unfolded protein response. Curr Opin Cell Biol 33, 67–73. Crossref, Medline, Google Scholar
• Walter P, Ron D (2011). The Unfolded Protein Response: From stress pathway to homeostatic regulation. Science 334, 1081–1086. Crossref, Medline, Google Scholar
• Wang DQ-H, Carey MC (2014). Therapeutic uses of animal biles in traditional Chinese medicine: An ethnopharmacological, biophysical chemical and medicinal review. World J Gastroenterol 20, 9952–9975. Crossref, Medline, Google Scholar
• Wang J, Pareja KA, Kaiser CA, Sevier CS (2014). Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress. Elife 3, e03496. Crossref, Medline, Google Scholar
• Wei H, Kim S-J, Zhang Z, Tsai P-C, Wisniewski KE, Mukherjee AB (2008). ER and oxidative stresses are common mediators of apoptosis in both neurodegenerative and non-neurodegenerative lysosomal storage disorders and are alleviated by chemical chaperones. Hum Mol Genet 17, 469–477. Crossref, Medline, Google Scholar
• Welch WJ, Randell Brown C (1996). Influence of molecular and chemical chaperones on protein folding. Cell Stress Chaperones 1, 109–115. Crossref, Medline, Google Scholar
• Winston JA, Theriot CM (2020). Diversification of host bile acids by members of the gut microbiota. Gut Microbes 11, 158–171. Crossref, Medline, Google Scholar
• Wu H, Ng BSH, Thibault G (2014). Endoplasmic reticulum stress response in yeast and humans. Biosci Rep 34, e00118. Crossref, Medline, Google Scholar
• Xie Q, Khaoustov VI, Chung CC, Sohn J, Krishnan B, Lewis DE, Yoffe B (2002). Effect of tauroursodeoxycholic acid on endoplasmic reticulum stress-induced caspase-12 activation. Hepatology 36, 592–601. Crossref, Medline, Google Scholar
• Xu M, Marsh HM, Sevier CS (2016). A Conserved cysteine within the ATPase domain of the endoplasmic reticulum chaperone BiP is necessary for a complete complement of BiP activities. J Mol Biol 428, 4168–4184. Crossref, Medline, Google Scholar
• Zhang L, Wang Y (2018). Tauroursodeoxycholic acid alleviates HO-induced oxidative stress and apoptosis via suppressing endoplasmic reticulum stress in neonatal rat cardiomyocytes. Dose Response 16, 1559325818782631. Crossref, Google Scholar
• Zhou Y, Doyen R, Lichtenberger LM (2009). The role of membrane cholesterol in determining bile acid cytotoxicity and cytoprotection of ursodeoxycholic acid. Biochim Biophys Acta 1788, 507–513. Crossref, Medline, Google Scholar