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Emerging Artemisinin (ART) resistance in Plasmodium falciparum (Pf) poses challenges for the discovery of novel drugs to tackle ART-resistant parasites. Concentrated efforts toward the ART resistance mechanism indicated a strong molecular link of ART resistance with upregulated expression of unfolded protein response pathways involving Prefoldins (PFDs). However, a complete characterization of PFDs as molecular players taking part in ART resistance mechanism, and discovery of small molecule inhibitors to block this process have not been identified to date. Here, we functionally characterized all Pf Prefoldin subunits (PFD1-6) and established a causative role played by PFDs in ART resistance by demonstrating their expression in intra-erythrocytic parasites along with their interactions with Kelch13 protein through immunoprecipitation coupled MS/MS analysis. Systematic biophysical interaction analysis between all subunits of PFDs revealed their potential to form a complex. The role of PFDs in ART resistance was confirmed in orthologous yeast PFD6 mutants, where PfPFD6 expression in yeast mutants reverted phenotype to ART resistance. We identified an FDA-approved drug “Biperiden” that restricts the formation of Prefoldin complex and inhibits its interaction with its key parasite protein substrates, MSP-1 and a-tubulin-I. Moreover, Biperiden treatment inhibits the parasite growth in ART-sensitive Pf3D7 and resistant Pf3D7k13R539T strains. Ring survival assays that are clinically relevant to analyze ART resistance in Pf3D7k13R539T parasites demonstrate the potency of BPD to inhibit the growth of survivor parasites. Overall, our study provides the first evidence of the role of PfPFDs in ART resistance mechanisms and opens new avenues for the management of resistant parasites. [ABSTRACT FROM AUTHOR]

The unfolded protein response pathways (UPR), autophagy, and compartmentalization of misfolded proteins into inclusion bodies are critical components of the protein quality control network. Among inclusion bodies, aggresomes are particularly intriguing due to their association with cellular survival, drug resistance, and aggresive cancer behavior. Aggresomes are molecular condensates formed when collapsed vimentin cages encircle misfolded proteins before final removal by autophagy. Yet significant gaps persist in the mechanisms governing aggresome formation and elimination in cancer cells. Understanding these mechanisms is crucial, especially considering the involvement of LC3A, a member of the MAP1LC3 family, which plays a unique role in autophagy regulation and has been reported to be epigenetically silenced in many cancers. Herein, we utilized the tetracycline-inducible expression of LC3A to investigate its role in choroid plexus carcinoma cells, which inherently exhibit the presence of aggresomes. Live cell imaging was employed to demonstrate the effect of LC3A expression on aggresome-positive cells, while SILAC-based proteomics identified LC3A-induced protein and pathway alterations. Our findings demonstrated that extended expression of LC3A is associated with cellular senescence. However, the obstruction of lysosomal degradation in this context has a deleterious effect on cellular viability. In response to LC3A-induced autophagy, we observed significant alterations in mitochondrial morphology, reflected by mitochondrial dysfunction and increased ROS production. Furthermore, LC3A expression elicited the activation of the PERK-eIF2α-ATF4 axis of the UPR, underscoring a significant change in the protein quality control network. In conclusion, our results elucidate that LC3A-mediated autophagy alters the protein quality control network, exposing a vulnerability in aggresome-positive cancer cells. [ABSTRACT FROM AUTHOR]

Tauopathies are neurodegenerative diseases caused by pathologic misfolded tau protein aggregation in the nervous system. Population studies implicate EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), better known as PERK (protein kinase R-like endoplasmic reticulum kinase), as a genetic risk factor in several tauopathies. PERK is a key regulator of intracellular proteostatic mechanisms-unfolded protein response and integrated stress response. Previous studies found that tauopathy-associated PERK variants encoded functional hypomorphs with reduced signaling in vitro. But, it remained unclear how altered PERK activity led to tauopathy. Here, we chemically or genetically modulated PERK signaling in cell culture models of tau aggregation and found that PERK pathway activation prevented tau aggregation, whereas inhibition exacerbated tau aggregation. In primary tauopathy patient brain tissues, we found that reduced PERK signaling correlated with increased tau neuropathology. We found that tauopathy-associated PERK variants targeted the endoplasmic reticulum luminal domain; and two of these variants damaged hydrogen bond formation. Our studies support that PERK activity protects against tau aggregation and pathology. This may explain why people carrying hypomorphic PERK variants have increased risk for developing tauopathies. Finally, our studies identify small-molecule augmentation of PERK signaling as an attractive therapeutic strategy to treat tauopathies by preventing tau pathology.

The stability of ribosomal DNA (rDNA) is maintained through transcriptional silencing by the NAD+ -dependent histone deacetylase Sir2 in Saccharomyces cerevisiae. Alongside proteostasis, rDNA stability is a crucial factor regulating the replicative lifespan of S. cerevisiae. The unfolded protein response (UPR) is induced by misfolding of proteins or an imbalance of membrane lipid composition and is responsible for degrading misfolded proteins and restoring endoplasmic reticulum (ER) membrane homeostasis. Recent investigations have suggested that the UPR can extend the replicative lifespan of yeast by enhancing protein quality control mechanisms, but the relationship between the UPR and rDNA stability remains unknown. In this study, we found that the deletion of ARV1, which encodes an ER protein of unknown molecular function, activates the UPR by inducing lipid bilayer stress. In arv1Δ cells, the UPR and the cell wall integrity pathway are activated independently of each other, and the high osmolarity glycerol (HOG) pathway is activated in a manner dependent on Ire1, which mediates the UPR. Activated Hog1 translocates the stress response transcription factor Msn2 to the nucleus, where it promotes the expression of nicotinamidase Pnc1, a wellknown Sir2 activator. Following Sir2 activation, rDNA silencing and rDNA stability are promoted. Furthermore, the loss of other ER proteins, such as Pmt1 or Bst1, and ER stress induced by tunicamycin or inositol depletion also enhance rDNA stability in a Hog1-dependent manner. Collectively, these findings suggest that the induction of the UPR enhances rDNA stability in S. cerevisiae by promoting the Msn2-Pnc1- Sir2 pathway in a Hog1-dependent manner. [ABSTRACT FROM AUTHOR]

The unfolded protein response is a mechanism aiming at restoring endoplasmic reticulum (ER) homeostasis and is likely involved in other adaptive pathways. The unfolded protein response is transduced by three proteins acting as sensors and triggering downstream signaling pathways. Among them, inositol-requiring enzyme 1 alpha (IRE1α) (referred to as IRE1 hereafter), an endoplasmic reticulum--resident type I transmembrane protein, exerts its function through both kinase and endoribonuclease activities, resulting in both X-box binding protein 1 mRNA splicing and RNA degradation (regulated ire1 dependent decay). An increasing number of studies have reported protein--protein interactions as regulators of these signaling mechanisms, and additionally, driving other noncanonical functions. In this review, we deliver evolutive and structural insights on IRE1 and further describe how this protein interaction network (interactome) regulates IRE1 signaling abilities or mediates other cellular processes through catalytic-independent mechanisms. Moreover, we focus on newly discovered targets of IRE1 kinase activity and discuss potentially novel IRE1 functions based on the nature of the interactome, thereby identifying new fields to explore regarding this protein's biological roles. [ABSTRACT FROM AUTHOR]

KDELR (Erd2 [ER retention defective 2] in yeasts) is a receptor protein that retrieves endoplasmic reticulum (ER)-resident proteins from the Golgi apparatus. However, the role of the KDELR-mediated ER-retrieval system in regulating cellular homeostasis remains elusive. Here, we show that the absence of Erd2 triggers the unfolded protein response (UPR) and enhances mitochondrial respiration and reactive oxygen species in an UPR-dependent manner in the fission yeast Schizosaccharomyces pombe. Moreover, we perform transcriptomic analysis and find that the expression of genes related to mitochondrial respiration and the tricarboxylic acid cycle is upregulated in a UPR-dependent manner in cells lacking Erd2. The increased mitochondrial respiration and reactive oxygen species production is required for cell survival in the absence of Erd2. Therefore, our findings reveal a novel role of the KDELR-Erd2-mediated ER-retrieval system in modulating mitochondrial functions and highlight its importance for cellular homeostasis in the fission yeast. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress and unfolded protein response are cells’ survival strategies to thwart disruption of proteostasis. Tumor cells are continuously being challenged by ER stress. The prion protein, PrP, normally a glycosylphosphatidylinositol (GPI)-anchored protein exists as a proPrP retaining its GPI-peptide signal sequence in human pancreatic ductal cell adenocarcinoma (PDAC). Higher abundance of pro-PrP indicates poorer prognosis in PDAC patients. The reason why PDAC cells express pro-PrP is unknown. Here, we report that persistent ER stress causes conversion of GPIanchored PrP to pro-PrP via a conserved ATF6–miRNA449c5p–PIGV axis. Mouse neurons and AsPC-1, a PDAC cell line, express GPI-anchored PrP. However, continuous culture of these cells with the ER stress inducers thapsigargin or brefeldin A results in the conversion of a GPI-anchored PrP to pro-PrP. Such a conversion is reversible; removal of the inducers allows the cells to re-express a GPI-anchored PrP. Mechanistically, persistent ER stress increases the abundance of an active ATF6, which increases the level of miRNA449c-5p (miR449c-5p). By binding the mRNA of PIGV at its 30 -UTRs, miR449c-5p suppresses the level of PIGV, a mannosyltransferase pivotal in the synthesis of the GPI anchor. Reduction of PIGV leads to disruption of the GPI anchor assembly, causing pro-PrP accumulation and enhancing cancer cell migration and invasion. The importance of ATF6–miR449c-5p–PIGV axis is recapitulated in PDAC biopsies as the higher levels of ATF6 and miR449c-5p and lower levels of PIGV are markers of poorer outcome for patients with PDAC. Drugs targeting this axis may prevent PDAC progression [ABSTRACT FROM AUTHOR]

Biological functions of the highly conserved ubiquitin-like protein 5 (UBL5) are not well understood. In Caenorhabditis elegans, UBL5 is induced under mitochondrial stress to mount the mitochondrial unfolded protein response (UPR). However, the role of UBL5 in the more prevalent endoplasmic reticulum (ER) stress-UPR in the mammalian system is unknown. In the present work, we demonstrated that UBL5 was an ER stress-responsive protein, undergoing rapid depletion in mammalian cells and livers of mice. The ER stress-induced UBL5 depletion was mediated by proteasome-dependent yet ubiquitin-independent proteolysis. Activation of the protein kinase R-like ER kinase arm of the UPR was essential and sufficient for inducing UBL5 degradation. RNA-Seq analysis of UBL5-regulated transcriptome revealed that multiple death pathways were activated in UBL5-silenced cells. In agreement with this, UBL5 knockdown induced severe apoptosis in culture and suppressed tumorigenicity of cancer cells in vivo. Furthermore, overexpression of UBL5 protected specifically against ER stress-induced apoptosis. These results identify UBL5 as a physiologically relevant survival regulator that is proteolytically depleted by the UPR-protein kinase R-like ER kinase pathway, linking ER stress to cell death. [ABSTRACT FROM AUTHOR]

The unfolded protein response (UPR) is sensitive to proteotoxic and membrane bilayer stress, both of which are sensed by the ER protein Ire1. When activated, Ire1 splices HAC1 mRNA, producing a transcription factor that targets genes involved in proteostasis and lipid metabolism, among others. The major membrane lipid phosphatidylcholine (PC) is subject to phospholipase-mediated deacylation, producing glycerophosphocholine (GPC), followed by reacylation of GPC through the PC deacylation/reacylation pathway (PC-DRP). The reacylation events occur via a two-step process catalyzed first by the GPC acyltransferase Gpc1, followed by acylation of the lyso-PC molecule by Ale1. However, whether Gpc1 is critical for ER bilayer homeostasis is unclear. Using an improved method for C14-choline-GPC radiolabeling, we first show that loss of Gpc1 results in abrogation of PC synthesis through PC-DRP and that Gpc1 colocalizes with the ER. We then probe the role of Gpc1 as both a target and an effector of the UPR. Exposure to the UPR-inducing compounds tunicamycin, DTT, and canavanine results in a Hac1-dependent increase in GPC1 message. Further, cells lacking Gpc1 exhibit increased sensitivity to those proteotoxic stressors. Inositol limitation, known to induce the UPR via bilayer stress, also induces GPC1 expression. Finally, we show that loss of GPC1 induces the UPR. A gpc1? mutant displays upregulation of the UPR in strains expressing a mutant form of Ire1 that is unresponsive to unfolded proteins, indicating that bilayer stress is responsible for the observed upregulation. Collectively, our data indicate an important role for Gpc1 in yeast ER bilayer homeostasis. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), which reduces levels of misfolded proteins. However, if ER homeostasis is not restored and the UPR remains chronically activated, cells undergo apoptosis. The UPR regulator, PKR-like endoplasmic reticulum kinase (PERK), plays an important role in promoting cell death when persistently activated; however, the underlying mechanisms are poorly understood. Here, we profiled the microRNA (miRNA) transcriptome in human cells exposed to ER stress and identified miRNAs that are selectively induced by PERK signaling. We found that expression of a PERK-induced miRNA, miR-483, promotes apoptosis in human cells. miR-483 induction was mediated by a transcription factor downstream of PERK, activating transcription factor 4 (ATF4), but not by the CHOP transcription factor. We identified the creatine kinase brain-type (CKB) gene, encoding an enzyme that maintains cellular ATP reserves through phosphocreatine production, as being repressed during the UPR and targeted by miR-483. We found that ER stress, selective PERK activation, and CKB knockdown all decrease cellular ATP levels, leading to increased vulnerability to ER stress-induced cell death. Our findings identify miR-483 as a downstream target of the PERK branch of the UPR. We propose that disruption of cellular ATP homeostasis through miR-483-mediated CKB silencing promotes ER stress-induced apoptosis.

In the mammalian heart, fetal cardiomyocytes proliferate prior to birth; however, they exit the cell cycle shortly after birth. Recent studies show that adult cardiomyocytes re-enters the cell cycle postinjury to promote cardiac regeneration. The endoplasmic reticulum (ER) orchestrates the production and assembly of different types of proteins, and a disruption in this machinery leads to the generation of ER stress, which activates the unfolded protein response. There is a very fine balance between ER stress-mediated protective and proapoptotic responses. T-box transcription factor 20 (Tbx20) promotes embryonic and adult cardiomyocyte proliferation postinjury to restore cardiac homeostasis. However, the function and regulatory interactions of Tbx20 in ER stress-induced cardiomyopathy have not yet been reported. We show here that ER stress upregulates Tbx20, which activates downstream bone morphogenetic protein 2 (Bmp2)-pSmad1/5/8 signaling to induce cardiomyocyte proliferation and limit apoptosis. However, augmenting ER stress reverses this protective response. We also show that increased expression of tbx20 during ER stress is mediated by the activating transcription factor 6 arm of the unfolded protein response. Cardiomyocyte-specific loss of Tbx20 results in decreased cardiomyocyte proliferation and increased apoptosis. Administration of recombinant Bmp2 protein during ER stress upregulates Tbx20 leading to augmented proliferation, indicating a feed-forward loop mechanism. In in vivo ER stress, as well as in diabetic cardiomyopathy, the activity of Tbx20 is increased with concomitant increased cardiomyocyte proliferation and decreased apoptosis. These data support a critical role of Tbx20-Bmp2 signaling in promoting cardiomyocyte survival during ER stress-induced cardiomyopathies. [ABSTRACT FROM AUTHOR]

As a central hub of cellular metabolism and signaling, the mitochondrion is a crucial organelle whose dysfunction can cause disease and whose activity is intimately connected to aging. We review how the mitochondrial network maintains proteomic integrity, how mitochondrial proteotoxic stress is communicated and resolved in the context of the entire cell, and how mitochondrial systems function in the context of organismal health and aging. A deeper understanding of how mitochondrial protein quality control mechanisms are coordinated across these distinct biological levels should help explain why these mechanisms fail with age and, ultimately, how routes to intervention might be attained.

Tauopathies are neurodegenerative diseases caused by pathologic misfolded tau protein aggregation in the nervous system. Population studies implicate EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), better known as PERK (protein kinase R-like endoplasmic reticulum kinase), as a genetic risk factor in several tauopathies. PERK is a key regulator of intracellular proteostatic mechanisms--unfolded protein response and integrated stress response. Previous studies found that tauopathy-associated PERK variants encoded functional hypomorphs with reduced signaling in vitro. But, it remained unclear how altered PERK activity led to tauopathy. Here, we chemically or genetically modulated PERK signaling in cell culture models of tau aggregation and found that PERK pathway activation prevented tau aggregation, whereas inhibition exacerbated tau aggregation. In primary tauopathy patient brain tissues, we found that reduced PERK signaling correlated with increased tau neuropathology. We found that tauopathy-associated PERK variants targeted the endoplasmic reticulum luminal domain; and two of these variants damaged hydrogen bond formation. Our studies support that PERK activity protects against tau aggregation and pathology. This may explain why people carrying hypomorphic PERK variants have increased risk for developing tauopathies. Finally, our studies identify small-molecule augmentation of PERK signaling as an attractive therapeutic strategy to treat tauopathies by preventing tau pathology. [ABSTRACT FROM AUTHOR]

Heart failure is associated with induction of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). The serine/threonine protein kinase/endoribonuclease IRE1α is a key protein in ER stress signal transduction. IRE1α activity can induce both protective UPR and apoptotic downstream signaling events, but the specific role for IRE1α activity in the heart is unknown. A major aim of this study was to characterize the specific contribution of IRE1α in cardiac physiology and pathogenesis. We used both cultured myocytes and a transgenic mouse line with inducible and cardiomyocyte-specific IRE1α overexpression as experimental models to achieve targeted IRE1α activation. IRE1α expression induced a potent but transient ER stress response in cardiomyocytes and did not cause significant effects in the intact heart under normal physiological conditions. Furthermore, the IRE1α-activated transgenic heart responding to pressure overload exhibited preserved function and reduced fibrotic area, associated with increased adaptive UPR signaling and with blunted inflammatory and pathological gene expression. Therefore, we conclude that IRE1α induces transient ER stress signaling and confers a protective effect against pressure overload-induced pathological remodeling in the heart. To our knowledge, this report provides first direct evidence of a specific and protective role for IRE1α in the heart and reveals an interaction between ER stress signaling and inflammatory regulation in the pathologically stressed heart.

Targeting of most integral membrane proteins to the endoplasmic reticulum is controlled by the signal recognition particle, which recognizes a hydrophobic signal sequence near the protein N terminus. Proper folding of these proteins is monitored by the unfolded protein response and involves protein degradation pathways to ensure quality control. Here, we identify a new pathway for quality control of major facilitator superfamily transporters that occurs before the first transmembrane helix, the signal sequence recognized by the signal recognition particle, is made by the ribosome. Increased rates of translation elongation of the N-terminal sequence of these integral membrane proteins can divert the nascent protein chains to the ribosome-associated complex and stress-seventy subfamily B chaperones. We also show that quality control of integral membrane proteins by ribosome-associated complex-stress-seventy subfamily B couples translation rate to the unfolded protein response, which has implications for understanding mechanisms underlying human disease and protein production in biotechnology.

Natural products constitute and significantly impact many current anti-cancer medical interventions. A subset of natural products induces injury processes in malignant cells that recruit and activate host immune cells to produce an adaptive anti-cancer immune response, a process known as immunogenic cell death. However, a challenge in the field is to delineate forms of cell death and injury that best promote durable antitumor immunity. Addressing this with a single-cell chemical biology natural product discovery platform, like multiplex activity metabolomics, would be especially valuable in human leukemia, where cancer cells are heterogeneous and may react differently to the same compounds. Herein, a new ten-color, fluorescent cell barcoding-compatible module measuring six immunogenic cell injury signaling readouts are as follows: DNA damage response (γH2AX), apoptosis (cCAS3), necroptosis (p-MLKL), mitosis (p-Histone H3), autophagy (LC3), and the unfolded protein response (p-EIF2α). A proof-of-concept screen was performed to validate functional changes in single cells induced by secondary metabolites with known mechanisms within bacterial extracts. This assay was then applied in multiplexed activity metabolomics to reveal an unexpected mammalian cell injury profile induced by the natural product narbomycin. Finally, the functional consequences of injury pathways on immunogenicity were compared with three canonical assays for immunogenic hallmarks, ATP, HMGB1, and calreticulin, to correlate secondary metabolite-induced cell injury profiles with canonical markers of immunogenic cell death. In total, this work demonstrated a new phenotypic screen for discovery of natural products that modulate injury response pathways that can contribute to cancer immunogenicity. [ABSTRACT FROM AUTHOR]

The concentration of Ca2+ in the endoplasmic reticulum (ER) is critically important for maintaining its oxidizing environment as well as for maintaining luminal ATP levels required for chaperone activity. Therefore, local luminal Ca2+ concentrations and the dynamic Ca2+ flux between the different subcellular compartments are tightly controlled. Influx of Ca2+ into the ER is enabled by a reductive shift, which opens the sarcoendoplasmic reticulum calcium transport ATPase pump, building the Ca2+ gradient across the ER membrane required for ATP import. Meanwhile, Ca2+ leakage from the ER has been reported to occur via the Sec61 translocon following protein translocation. In this review, we provide an overview of the complex regulation of Ca2+ homeostasis, Ca2+ flux between subcellular compartments, and the cellular stress response (the unfolded protein response) induced upon dysregulated luminal Ca2+ metabolism. We also provide insight into the structure and gating mechanism at the Sec61 translocon and examine the role of ER-resident cochaperones in assisting the central ER-resident chaperone BiP in the control of luminal Ca2+ concentrations. [ABSTRACT FROM AUTHOR]

Inositol-requiring enzyme 1 (IRE1) is an evolutionarily conserved sensor of endoplasmic reticulum (ER) stress and mediates a key branch of the unfolded protein response in eukaryotic cells. It is an ER-resident transmembrane protein that possesses Ser/Thr protein kinase and endoribonuclease (RNase) activities in its cytoplasmic region. IRE1 is activated through dimerization/oligomerization and autophosphorylation at multiple sites, acting through its RNase activity to restore the functional capacity of the ER. However, it remains poorly defined in vivo how the autophosphorylation events of endogenous IRE1 govern its dynamic activation and functional output. Here, we generated a mouse model harboring a S724A knock-in mutation (Ern1S724A/S724A) and investigated the importance of phosphorylation at Ser724 within the kinase activation loop of murine IRE1α. We found that in mouse embryonic fibroblast cells and in primary hepatocytes, S724A mutation resulted in markedly reduced IRE1α autophosphorylation in parallel with blunted activation of its RNase activity to catalyze X-box binding protein 1 (Xbp1) mRNA splicing. Furthermore, ablation of IRE1α phosphorylation at Ser724 exacerbated ER stress–induced hepatic steatosis in tunicamycin- treated Ern1S724A/S724A mice. This was accompanied by significantly decreased hepatic production of spliced XBP1 protein but increased CCAAT-enhancer–binding protein homologous protein (CHOP) level, along with suppressed expression of key metabolic regulators of fatty acid β-oxidation and lipid secretion. These results demonstrate a critical role of phosphorylation at Ser724 of IRE1α in dynamically controlling its kinase activity, and thus its autophosphorylation state, which is coupled to activation of its RNase activity in counteracting hepatic steatosis under ER stress conditions. [ABSTRACT FROM AUTHOR]

The unfolded protein response (UPR) is an adaptation mechanism activated to resolve transient accumulation of unfolded/misfolded proteins in the endoplasmic reticulum. Failure to resolve the transient accumulation of such proteins results in UPR-mediated programmed cell death. Loss of tumor suppressor gene or oncogene addiction in cancer cells can result in sustained higher basal UPR levels; however, it is not clear if these higher basal UPR levels in cancer cells can be exploited as a therapeutic strategy. We hypothesized that covalent modification of surface-exposed cysteine (SEC) residues could simulate unfolded/misfolded proteins to activate the UPR, and that higher basal UPR levels in cancer cells would provide the necessary therapeutic window. To test this hypothesis, here we synthesized analogs that can covalently modify multiple SEC residues and evaluated them as UPR activators. We identified a spirocyclic dimer, SpiD7, and evaluated its effects on UPR activation signals, that is, XBP1 splicing, phosphorylation of eIF2α, and a decrease in ATF 6 levels, in normal and cancer cells, which were further confirmed by RNA-Seq analyses. We found that SpiD7 selectively induced caspase-mediated apoptosis in cancer cells, whereas normal cells exhibited robust XBP1 splicing, indicating adaptation to stress. Furthermore, SpiD7 inhibited the growth of high-grade serous carcinoma cell lines ~3-15-fold more potently than immortalized fallopian tube epithelial (paired normal control) cells and reduced clonogenic growth of high-grade serous carcinoma cell lines. Our results suggest that induction of the UPR by covalent modification of SEC residues represents a cancer cell vulnerability and can be exploited to discover novel therapeutics. [ABSTRACT FROM AUTHOR]

Although several proteasome subunits have been shown to bind ubiquitin (Ub) chains, many ubiquitylated substrates also associate with 26S proteasomes via "shuttling factors." Unlike the well-studied yeast shuttling factors Rad23 and Dsk2, vertebrate homologs Ddi2 and Ddi1 lack a Ub-associated domain; therefore, it is unclear how they bind Ub. Here, we show that deletion of Ddi2 leads to the accumulation of Ub conjugates with K11/K48 branched chains. We found using affinity copurifications that Ddi2 binds Ub conjugates through its Ub-like domain, which is also required for Ddi2 binding to proteasomes. Furthermore, in cell extracts, adding Ub conjugates increased the amount of Ddi2 associated with proteasomes, and adding Ddi2 increased the binding of Ub conjugates to purified proteasomes. In addition, Ddi2 also contains a retroviral protease domain with undefined cellular roles. We show that blocking the endoprotease activity of Ddi2 either genetically or with the HIV protease inhibitor nelfinavir increased its binding to Ub conjugates but decreased its binding to proteasomes and reduced subsequent protein degradation by proteasomes leading to further accumulation of Ub conjugates. Finally, nelfinavir treatment required Ddi2 to induce the unfolded protein response. Thus, Ddi2 appears to function as a shuttling factor in endoplasmic reticulum-associated protein degradation and delivers K11/K48-ubiquitylated proteins to the proteasome. We conclude that the protease activity of Ddi2 influences this shuttling factor activity, promotes protein turnover, and helps prevent endoplasmic reticulum stress, which may explain nelfinavir's ability to enhance cell killing by proteasome inhibitors. [ABSTRACT FROM AUTHOR]

Endocrine-therapy-resistant estrogen receptor-positive (ER+) breast cancer cells often exhibit an augmented capacity to maintain endoplasmic reticulum (EnR) homeostasis under adverse conditions. Oncoprotein hepatitis B X-interacting protein (HBXIP) is a known transcriptional coactivator that promotes cancer development. However, it is unclear whether HBXIP participates in maintaining EnR homeostasis and promoting drug resistance in ER+ breast cancer. Here, we report that tamoxifen-resistant (TmaR) breast cancer cells exhibit increased expression of HBXIP, which acts as an inactivator of the unfolded protein response (UPR) to diminish tamoxifeninduced EnR stress. We show that HBXIP deficiency promotes EnR-associated degradation, enhances UPR-element reporter activity and cellular oxidative stress, and ultimately attenuates the growth of TmaR cells in vitro and in vivo. Mechanistically, we demonstrate that HBXIP acts as a chaperone of UPR transducer inositol-requiring enzyme 1a and diminishes production of reactive oxygen species (ROS) in TamR breast cancer cells. Upon loss of HBXIP expression, tamoxifen treatment hyperactivates IRE1α and its downstream proapoptotic pathways and simultaneously induces accumulation of intracellular ROS. This elevated ROS programmatically activates the other two branches of the UPR, mediated by PKR-like ER kinase and activating transcription factor 6α. Clinical investigations and Kaplan-Meier plotter analysis revealed that HBXIP is highly expressed in TamR breast cancer tissues. Furthermore, reinforced HBXIP expression is associated with a high recurrence and poor relapse-free survival rates in tamoxifen monotherapy ER+ breast cancer patients. These findings indicate that HBXIP is a regulator of EnR homeostasis and a potential target for TamR breast cancer therapy. [ABSTRACT FROM AUTHOR]

Current immunosuppressive strategies in organ transplantation rely on calcineurin inhibitors cyclosporine A (CsA) or tacrolimus (Tac). Both drugs are nephrotoxic, but CsA has been associated with greater renal damage than Tac. CsA inhibits calcineurin by forming complexes with cyclophilins, whose chaperone function is essential for proteostasis. We hypothesized that stronger toxicity of CsA may be related to suppression of cyclophilins with ensuing endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in kidney epithelia. Effects of CsA and Tac (10 mM for 6 h each) were compared in cultured human embryonic kidney 293 (HEK 293) cells, primary human renal proximal tubule (PT) cells, freshly isolated rat PTs, and knockout HEK 293 cell lines lacking the critical ER stress sensors, protein kinase RNA-like ER kinase or activating transcription factor 6 (ATF6). UPR was evaluated by detection of its key components. Compared with Tac treatment, CsA induced significantly stronger UPR in native cultured cells and isolated PTs. Evaluation of proapoptotic and antiapoptotic markers suggested an enhanced apoptotic rate in CsA-treated cells compared with Tac-treated cells as well. Similar to CsA treatment, knockdown of cyclophilin A or B by siRNA caused proapoptotic UPR, whereas application of the chemical chaperones tauroursodeoxycholic acid or 4-phenylbutyric acid alleviated CsA-induced UPR. Deletion of protein kinase RNA-like ER kinase or ATF6 blunted CsA-induced UPR as well. In summary, inhibition of cyclophilin chaperone function with ensuing ER stress and proapoptotic UPR aggravates CsA toxicity, whereas pharmacological modulation of UPR bears potential to alleviate renal side effects of CsA. [ABSTRACT FROM AUTHOR]

Type 2 diabetes is a metabolic disorder associated with abnormal glucose homeostasis and is characterized by intrinsic defects in β-cell function and mass. Trimethylguanosine synthase 1 (TGS1) is an evolutionarily conserved enzyme that methylates small nuclear and nucleolar RNAs and that is involved in pre-mRNA splicing, transcription, and ribosome production. However, the role of TGS1 in β-cells and glucose homeostasis had not been explored. Here, we show that TGS1 is upregulated by insulin and upregulated in islets of Langerhans from mice exposed to a high-fat diet and in human β-cells from type 2 diabetes donors. Using mice with conditional (βTGS1KO) and inducible (MIP-CreERT-TGS1KO) TGS1 deletion, we determined that TGS1 regulates β-cell mass and function. Using unbiased approaches, we identified a link between TGS1 and endoplasmic reticulum stress and cell cycle arrest, as well as and how TGS1 regulates β-cell apoptosis. We also found that deletion of TGS1 results in an increase in the unfolded protein response by increasing XBP-1, ATF-4, and the phosphorylation of eIF2α, in addition to promoting several changes in cell cycle inhibitors and activators such as p27 and Cyclin D2. This study establishes TGS1 as a key player regulating β-cell mass and function. We propose that these observations can be used as a stepping-stone for the design of novel strategies focused on TGS1 as a therapeutic target for the treatment of diabetes. [ABSTRACT FROM AUTHOR]

Persistent inactivity promotes skeletal muscle atrophy, marked by mitochondrial aberrations that affect strength, mobility, and metabolic health leading to the advancement of disease. Mitochondrial quality control (MQC) pathways include biogenesis (synthesis), mitophagy/lysosomal turnover, and the mitochondrial unfolded protein response, which serve to maintain an optimal organelle network. Tumor suppressor p53 has been implicated in regulating muscle mitochondria in response to cellular stress; however, its role in the context of muscle disuse has yet to be explored, and whether p53 is necessary for MQC remains unclear. To address this, we subjected p53 muscle-specific KO (mKO) and WT mice to unilateral denervation. Transcriptomic and pathway analyses revealed dysregulation of pathways pertaining to mitochondrial function, and especially turnover, in mKO muscle following denervation. Protein and mRNA data of the MQC pathways indicated activation of the mitochondrial unfolded protein response and mitophagy-lysosome systems along with reductions in mitochondrial biogenesis and content in WT and mKO tissue following chronic denervation. However, p53 ablation also attenuated the expression of autophagy-mitophagy machinery, reduced autophagic flux, and enhanced lysosomal dysfunction. While similar reductions in mitochondrial biogenesis and content were observed between genotypes, MQC dysregulation exacerbated mitochondrial dysfunction in mKO fibers, evidenced by elevated reactive oxygen species. Moreover, acute experiments indicate that p53 mediates the expression of transcriptional regulators of MQC pathways as early as 1 day following denervation. Together, our data illustrate exacerbated mitochondrial dysregulation with denervation stress in p53 mKO tissue, thus indicating that p53 contributes to organellar maintenance via regulation of MQC pathways during muscle atrophy. [ABSTRACT FROM AUTHOR]

Hepatic ischemia/reperfusion (I/R) injury is an inflammation-mediated process arising from ischemia/reperfusion-elicited stress in multiple cell types, causing liver damage during surgical procedures and often resulting in liver failure. Endoplasmic reticulum (ER) stress triggers the activation of the unfolded protein response (UPR) and is implicated in tissue injuries, including hepatic I/R injury. However, the cellular mechanism that links the UPR signaling to local inflammatory responses during hepatic I/R injury remains largely obscure. Here, we report that IRE1α, a critical ER-resident transmembrane signal transducer of the UPR, plays an important role in promoting Kupffer-cell-mediated liver inflammation and hepatic I/R injury. Utilizing a mouse model in which IRE1α is specifically ablated in myeloid cells, we found that abrogation of IRE1α markedly attenuated necrosis and cell death in the liver, accompanied by reduced neutrophil infiltration and liver inflammation following hepatic I/R injury. Mechanistic investigations in mice as well as in primary Kupffer cells revealed that loss of IRE1α in Kupffer cells not only blunted the activation of the NLRP3 inflammasome and IL-1β production, but also suppressed the expression of the inducible nitric oxide synthase (iNos) and proinflammatory cytokines. Moreover, pharmacological inhibition of IRE1α's RNase activity was able to attenuate inflammasome activation and iNos expression in Kupffer cells, leading to alleviation of hepatic I/R injury. Collectively, these results demonstrate that Kupffer cell IRE1α mediates local inflammatory damage during hepatic I/R injury. Our findings suggest that IRE1α RNase activity may serve as a promising target for therapeutic treatment of ischemia/reperfusion-associated liver inflammation and dysfunction. [ABSTRACT FROM AUTHOR]

Protein disulfide isomerase-A1 (PDIA1) is a master regulator of oxidative protein folding and proteostasis in the endoplasmic reticulum (ER). However, PDIA1 can reach the extracellular space, impacting thrombosis and other pathophysiological phenomena. Whether PDIA1 is externalized via passive release or active secretion is not known. To investigate how PDIA1 negotiates its export, we generated a tagged variant that undergoes N-glycosylation in the ER (Glyco-PDIA1). Addition of N-glycans does not alter its enzymatic functions. Upon either deletion of its KDEL ER-localization motif or silencing of KDEL receptors, Glyco-PDIA1 acquires complex glycans in the Golgi and is secreted. In control cells, however, Glyco-PDIA1 is released with endoglycosidase-H sensitive glycans, implying that it does not follow the classical ER-Golgi route nor does it encounter glycanases in the cytosol. Extracellular Glyco-PDIA1 is more abundant than actin, lactate dehydrogenase, or other proteins released by damaged or dead cells, suggesting active transport through a Golgi-independent route. The strategy we describe herein can be extended to dissect how select ER-residents reach the extracellular space., Competing Interests: Conflicts of interest The authors declare no conflict of interest with contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Fic domain-containing AMP transferases (fic AMPylases) are conserved enzymes that catalyze the covalent transfer of AMP to proteins. This posttranslational modification regulates the function of several proteins, including the ER-resident chaperone Grp78/BiP. Here we introduce a mouse FICD (mFICD) AMPylase knockout mouse model to study fic AMPylase function in vertebrates. We find that mFICD deficiency is well tolerated in unstressed mice. We also show that mFICDdeficient mouse embryonic fibroblasts are depleted of AMPylated proteins. mFICD deletion alters protein synthesis and secretion in splenocytes, including that of IgM, an antibody secreted early during infections, and the proinflammatory cytokine IL-1β, without affecting the unfolded protein response. Finally, we demonstrate that visual nonspatial shortterm learning is stronger in old mFICD−/− mice than in wildtype controls while other measures of cognition, memory, and learning are unaffected. Together, our results suggest a role for mFICD in adaptive immunity and neuronal plasticity in vivo. [ABSTRACT FROM AUTHOR]

The unfolded protein response plays an evolutionarily conserved role in homeostasis, and its dysregulation often leads to human disease, including diabetes and cancer. IRE1a is a major transducer that conveys endoplasmic reticulum stress via biochemical signals, yet major gaps persist in our understanding of how the detection of stress is converted to one of several molecular outcomes. It is known that, upon sensing unfolded proteins via its endoplasmic reticulum luminal domain, IRE1a dimerizes and then oligomerizes (often visualized as clustering). Once assembled, the kinase domain trans-autophosphorylates a neighboring IRE1a, inducing a conformational change that activates the RNase effector domain. However, the full details of how the signal is transmitted are not known. Here, we describe a previously unrecognized role for helix aK, located between the kinase and RNase domains of IRE1a, in conveying this critical conformational change. Using constructs containing mutations within this interdomain helix, we show that distinct substitutions affect oligomerization, kinase activity, and the RNase activity of IRE1a differentially. Furthermore, using both biochemical and computational methods, we found that different residues at position 827 specify distinct conformations at distal sites of the protein, such as in the RNase domain. Of importance, an RNase-inactive mutant, L827P, can still dimerize with wildtype monomers, but this mutation inactivates the wildtype molecule and renders leukemic cells more susceptible to stress. We surmise that helix aK is a conduit for the activation of IRE1a in response to stress. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus 2 (SARSCoV-2), the causative agent of the COVID-19 global pandemic, utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) for viral entry. However, other host factors might also play important roles in SARS-CoV-2 infection, providing new directions for antiviral treatments. GRP78 is a stress-inducible chaperone important for entry and infectivity for many viruses. Recent molecular docking analyses revealed putative interaction between GRP78 and the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (SARS-2-S). Here we report that GRP78 can form a complex with SARS-2-S and ACE2 on the surface and at the perinuclear region typical of the endoplasmic reticulum in VeroE6-ACE2 cells and that the substratebinding domain of GRP78 is critical for this interaction. In vitro binding studies further confirmed that GRP78 can directly bind to the RBD of SARS-2-S and ACE2. To investigate the role of GRP78 in this complex, we knocked down GRP78 in VeroE6-ACE2 cells. Loss of GRP78 markedly reduced cell surface ACE2 expression and led to activation of markers of the unfolded protein response. Treatment of lung epithelial cells with a humanized monoclonal antibody (hMAb159) selected for its safe clinical profile in preclinical models depleted cell surface GRP78 and reduced cell surface ACE2 expression, as well as SARS-2-S-driven viral entry and SARS-CoV-2 infection in vitro. Our data suggest that GRP78 is an important host auxiliary factor for SARS-CoV-2 entry and infection and a potential target to combat this novel pathogen and other viruses that utilize GRP78 in combination therapy. [ABSTRACT FROM AUTHOR]

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-stress-regulated protein exhibiting cytoprotective properties through a poorly understood mechanism in various in vitro and in vivo models of neuronal and non-neuronal damage. Although initially characterized as a secreted neurotrophic factor for midbrain dopamine neurons, MANF has recently gained more interest for its intracellular role in regulating the ER homeostasis, including serving as a cofactor of the chaperone glucose-regulated protein 78 (GRP78). We aimed for a better understanding of the neuroprotective mechanisms of MANF. Here we show for the first time thatMANFpromotes the survival of ER-stressed neurons in vitro as a general unfolded protein response (UPR) regulator, affecting severalUPRpathways simultaneously. Interestingly, MANF does not affect naïve neurons. We hypothesize that MANF regulates UPR signaling toward a mode more compatible with neuronal survival. Screening of MANF interacting proteins from two mammalian cell lines revealed a conserved interactome of 15 proteins including several ER chaperones such as GRP78, GRP170, protein disulfide isomerase family A member 1, and protein disulfide isomerase family A member 6. Further characterization confirmed previously published finding that MANF is a cofactor of GRP78 interacting with its nucleotide binding domain. Using microscale thermophoresis and nuclear magnetic resonance spectroscopy, we discovered that MANF is an ATP binding protein and that ATP blocks the MANF-GRP78 interaction. Interestingly, functional analysis of the antiapoptotic properties of MANF mutants in cultured neurons revealed divergent roles of MANF as a GRP78 cofactor and as an antiapoptotic regulator of UPR. We conclude that the co-factor type interaction with GRP78 is dispensable for the survivalpromoting activity of MANF in neurons. [ABSTRACT FROM AUTHOR]

Large cytosolic protein aggregates are removed by two main cellular processes, autophagy and the ubiquitin-proteasome system, and defective clearance of these protein aggregates results in proteotoxicity and cell death. Recently, we found that the eIF2a kinase heme-regulated inhibitory (HRI) induced a cytosolic unfolded protein response to prevent aggregation of innate immune signalosomes, but whether HRI acts as a general sensor of proteotoxicity in the cytosol remains unclear. Here we show that HRI controls autophagy to clear cytosolic protein aggregates when the ubiquitin-proteasome system is inhibited. We further report that silencing the expression of HRI resulted in decreased levels of BAG3 and HSPB8, two proteins involved in chaperone-assisted selective autophagy, suggesting that HRI may control proteostasis in the cytosol at least in part through chaperone-assisted selective autophagy. Moreover, knocking down the expression of HRI resulted in cytotoxic accumulation of overexpressed a-synuclein, a protein known to aggregate in Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In agreement with these data, protein aggregate accumulation and microglia activation were observed in the spinal cord white matter of 7-month-old Hri-/- mice as compared with Hri+/+ littermates. Moreover, aged Hri-/- mice showed accumulation of misfolded a-synuclein in the lateral collateral pathway, a region of the sacral spinal cord horn that receives visceral sensory afferents from the bladder and distal colon, a pathological feature common to a-synucleinopathies in humans. Together, these results suggest that HRI contributes to a general cytosolic unfolded protein response that could be leveraged to bolster the clearance of cytotoxic protein aggregates. [ABSTRACT FROM AUTHOR]

Missense mutations in ATP1A3, the α3 isoform of Na,KATPase, cause neurological phenotypes that differ greatly in symptoms and severity. A mechanistic basis for differences is lacking, but reduction of activity alone cannot explain them. Isogenic cell lines with endogenous α1 and inducible exogenous α3 were constructed to compare mutation properties. Na,K-ATPase is made in the endoplasmic reticulum (ER), but the glycan-free catalytic α subunit complexes with glycosylated β subunit in the ER to proceed through Golgi and post-Golgi trafficking. We previously observed classic evidence of protein misfolding in mutations with severe phenotypes: differences in ER retention of endogenous β1 subunit, impaired trafficking of α3, and cytopathology, suggesting that they misfold during biosynthesis. Here we tested two mutations associated with different phenotypes: D923N, which has a median age of onset of hypotonia or dystonia at 3 years, and L924P, with severe infantile epilepsy and profound impairment. Misfolding during biosynthesis in the ER activates the unfolded protein response, a multiarmed program that enhances protein folding capacity, and if that fails, triggers apoptosis. L924P showed more nascent protein retention in ER than D923N; more ER-associated degradation of α3 (ERAD); larger differences in Na,K-ATPase subunit distributions among subcellular fractions; and greater inactivation of eIF2α, a major defensive step of the unfolded protein response. In L924P there was also altered subcellular distribution of endogenous α1 subunit, analogous to a dominant negative effect. Both mutations showed pro-apoptotic sensitization by reduced phosphorylation of BAD. Encouragingly, however, 4-phenylbutyrate, a pharmacological corrector, reduced L924P ER retention, increased α3 expression, and restored morphology. [ABSTRACT FROM AUTHOR]

Variable number of tandem repeat (VNTR) sequences in the genome can have functional consequences that contribute to human disease. This is the case for the CEL gene, which is specifically expressed in pancreatic acinar cells and encodes the digestive enzyme carboxyl ester lipase. Rare single-base deletions (DELs) within the first (DEL1) or fourth (DEL4) VNTR segment of CEL cause maturity-onset diabetes of the young, type 8 (MODY8), an inherited disorder characterized by exocrine pancreatic dysfunction and diabetes. Studies on the DEL1 variant have suggested that MODY8 is initiated by CEL protein misfolding and aggregation. However, it is unclear how the position of single-base deletions within the CEL VNTR affects pathogenic properties of the protein. Here, we investigated four naturally occurring CEL variants, arising from single-base deletions in different VNTR segments (DEL1, DEL4, DEL9, and DEL13). When the four variants were expressed in human embryonic kidney 293 cells, only DEL1 and DEL4 led to significantly reduced secretion, increased intracellular aggregation, and increased endoplasmic reticulum stress compared with normal CEL protein. The level of Oglycosylation was affected in all DEL variants. Moreover, all variants had enzymatic activity comparable with that of normal CEL. We conclude that the longest aberrant protein tails, resulting from single-base deletions in the proximal VNTR segments, have highest pathogenic potential, explaining why DEL1 and DEL4 but not DEL9 and DEL13 have been observed in patients with MODY8. These findings further support the view that CEL mutations cause pancreatic disease through protein misfolding and proteotoxicity, leading to endoplasmic reticulum stress and activation of the unfolded protein response. [ABSTRACT FROM AUTHOR]

Lipid transfer proteins of the Ups1/PRELID1 family facilitate the transport of phospholipids across the intermembrane space of mitochondria in a lipid-specific manner. Heterodimeric complexes of yeast Ups1/Mdm35 or human PRELID1/TRIAP1 shuttle phosphatidic acid (PA) mainly synthesized in the endoplasmic reticulum (ER) to the inner membrane, where it is converted to cardiolipin (CL), the signature phospholipid of mitochondria. Loss of Ups1/PRELID1 proteins impairs the accumulation of CL and broadly affects mitochondrial structure and function. Unexpectedly and unlike yeast cells lacking the CL synthase Crd1, Ups1-deficient yeast cells exhibit glycolytic growth defects, pointing to functions of Ups1- mediated PA transfer beyond CL synthesis. Here, we show that the disturbed intramitochondrial transport of PA in ups1Δ cells leads to altered unfolded protein response (UPR) and mTORC1 signaling, independent of disturbances in CL synthesis. The impaired flux of PA into mitochondria is associated with the increased synthesis of phosphatidylcholine and a reduced phosphatidylethanolamine/phosphatidylcholine ratio in the ER of ups1Δ cells which suppresses the UPR. Moreover, we observed inhibition of target of rapamycin complex 1 (TORC1) signaling in these cells. Activation of either UPR by ER protein stress or of TORC1 signaling by disruption of its negative regulator, the Seh1-associated complex inhibiting TORC1 complex, increased cytosolic protein synthesis, and restored glycolytic growth of ups1Δ cells. These results demonstrate that PA influx into mitochondria is required to preserve ER membrane homeostasis and that its disturbance is associated with impaired glycolytic growth and cellular stress signaling. [ABSTRACT FROM AUTHOR]

DNA damage triggers the cellular adaptive response to arrest proliferation and repair DNA damage; when damage is too severe to be repaired, apoptosis is initiated to prevent the spread of genomic insults. However, how cells endure DNA damage to maintain cell function remains largely unexplored. By using Caenorhabditis elegans as a model, we report that DNA damage elicits cell maintenance programs, including the unfolded protein response of the endoplasmic reticulum (UPRER). Mechanistically, sublethal DNA damage unexpectedly suppresses apoptotic genes in C. elegans, which in turn increases the activity of the inositol-requiring enzyme 1/X-box binding protein 1 (IRE-1/XBP-1) branch of the UPRER by elevating unsaturated phosphatidylcholine. In addition, UPRER activation requires silencing of the lipid regulator skinhead-1 (SKN-1). DNA damage suppresses SKN-1 activity to increase unsaturated phosphatidylcholine and activate UPRER. These findings reveal the UPRER activation as an organismal adaptive response that is important to maintain cell function during DNA damage. [ABSTRACT FROM AUTHOR]

The stability of ribosomal DNA (rDNA) is maintained through transcriptional silencing by the NAD + -dependent histone deacetylase Sir2 in Saccharomyces cerevisiae. Alongside proteostasis, rDNA stability is a crucial factor regulating the replicative lifespan of S. cerevisiae. The unfolded protein response (UPR) is induced by misfolding of proteins or an imbalance of membrane lipid composition and is responsible for degrading misfolded proteins and restoring endoplasmic reticulum (ER) membrane homeostasis. Recent investigations have suggested that the UPR can extend the replicative lifespan of yeast by enhancing protein quality control mechanisms, but the relationship between the UPR and rDNA stability remains unknown. In this study, we found that the deletion of ARV1, which encodes an ER protein of unknown molecular function, activates the UPR by inducing lipid bilayer stress. In arv1Δ cells, the UPR and the cell wall integrity pathway are activated independently of each other, and the high osmolarity glycerol (HOG) pathway is activated in a manner dependent on Ire1, which mediates the UPR. Activated Hog1 translocates the stress response transcription factor Msn2 to the nucleus, where it promotes the expression of nicotinamidase Pnc1, a well-known Sir2 activator. Following Sir2 activation, rDNA silencing and rDNA stability are promoted. Furthermore, the loss of other ER proteins, such as Pmt1 or Bst1, and ER stress induced by tunicamycin or inositol depletion also enhance rDNA stability in a Hog1-dependent manner. Collectively, these findings suggest that the induction of the UPR enhances rDNA stability in S. cerevisiae by promoting the Msn2-Pnc1-Sir2 pathway in a Hog1-dependent manner., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

KDELR (Erd2 [ER retention defective 2] in yeasts) is a receptor protein that retrieves endoplasmic reticulum (ER)-resident proteins from the Golgi apparatus. However, the role of the KDELR-mediated ER-retrieval system in regulating cellular homeostasis remains elusive. Here, we show that the absence of Erd2 triggers the unfolded protein response (UPR) and enhances mitochondrial respiration and reactive oxygen species in an UPR-dependent manner in the fission yeast Schizosaccharomyces pombe. Moreover, we perform transcriptomic analysis and find that the expression of genes related to mitochondrial respiration and the tricarboxylic acid cycle is upregulated in a UPR-dependent manner in cells lacking Erd2. The increased mitochondrial respiration and reactive oxygen species production is required for cell survival in the absence of Erd2. Therefore, our findings reveal a novel role of the KDELR-Erd2-mediated ER-retrieval system in modulating mitochondrial functions and highlight its importance for cellular homeostasis in the fission yeast., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Degradation by proteasomes involves coupled translocation and unfolding of its protein substrates. Six distinct but paralogous proteasome ATPase proteins, Rpt1 to -6, form a heterohexameric ring that acts on substrates. An axially positioned loop (Ar-Φ loop) moves in concert with ATP hydrolysis, engages substrate, and propels it into a proteolytic chamber. The aromatic (Ar) residue of the Ar-Φ loop in all six Rpts of S. cerevisiae is tyrosine; this amino acid is thought to have important functional contacts with substrate. Six yeast strains were constructed and characterized in which Tyr was individually mutated to Ala. The mutant cells were viable and had distinct phenotypes. rpt3, rpt4, and rpt5 Tyr/Ala mutants, which cluster on one side of the ATPase hexamer, were substantially impaired in their capacity to degrade substrates. In contrast, rpt1, rpt2, and rpt6 mutants equaled or exceeded wild type in degradation activity. However, rpt1 and rpt6 mutants had defects that limited cell growth or viability under conditions that stressed the ubiquitin proteasome system. In contrast, the rpt3 mutant grew faster than wild type and to a smaller size, a defect that has previously been associated with misregulation of G1 cyclins. This rpt3 phenotype probably results from altered degradation of cell cycle regulatory proteins. Finally, mutation of five of the Rpt subunits increased proteasome ATPase activity, implying bidirectional coupling between the Ar-Φ loop and the ATP hydrolysis site. The present observations assign specific functions to individual Rpt proteins and provide insights into the diverse roles of the axial loops of individual proteasome ATPases.

The unfolded protein response (UPR) plays a central role in regulating endoplasmic reticulum(ER) and global cellular physiology in response to pathologic ER stress. The UPR is comprised of three signaling pathways activated downstream of the ER membrane proteins IRE1, ATF6, and PERK. Once activated, these proteins initiate transcriptional and translational signaling that functions to alleviate ER stress, adapt cellular physiology, and dictate cell fate. Imbalances in UPR signaling are implicated in the pathogenesis of numerous, etiologicallydiverse diseases, including many neurodegenerative diseases, protein misfolding diseases, diabetes, ischemic disorders, and cancer. This has led to significant interest in establishing pharmacologic strategies to selectively modulate IRE1, ATF6, or PERK signaling to both ameliorate pathologic imbalances in UPR signaling implicated in these different diseases and define the importance of the UPR in diverse cellular and organismal contexts. Recently, there has been significant progress in the identification and characterization of UPR modulating compounds, providing new opportunities to probe the pathologic and potentially therapeutic implications of UPR signaling in human disease. Here, we describe currently available UPR modulating compounds, specifically highlighting the strategies used for their discovery and specific advantages and disadvantages in their application for probing UPR function. Furthermore, we discuss lessons learned from the application of these compounds in cellular and in vivo models to identify favorable compound properties that can help drive the further translational development of selective UPR modulators for human disease. [ABSTRACT FROM AUTHOR]

Autosomal recessive mutations in the galactosidase β1 (GLB1) gene cause lysosomal β-gal deficiency, resulting in accumulation of galactose-containing substrates and onset of the progressive and fatal neurodegenerative lysosomal storage disease, GM1 gangliosidosis. Here, an enzyme replacement therapy (ERT) approach in fibroblasts from GM1 gangliosidosis patients with recombinant human β-gal (rhβ-gal) produced in Chinese hamster ovary cells enabled direct and precise rhβ-gal delivery to acidified lysosomes. A single, low dose (3 nM) of rhβ-gal was sufficient for normalizing β-gal activity and mediating substrate clearance for several weeks. We found that rhβ-gal uptake by the fibroblasts is dose-dependent and saturable and can be competitively inhibited by mannose 6-phosphate, suggesting cation-independent, mannose 6-phosphate receptor–mediated endocytosis from the cell surface. A single intracerebroventricularly (ICV) administered dose of rhβ-gal (100 μg) resulted in broad bilateral biodistribution of rhβ-gal to critical regions of pathology in a mouse model of GM1 gangliosidosis. Weekly ICV dosing of rhβ-gal for 8 weeks substantially reduced brain levels of ganglioside and oligosaccharide substrates and reversed well-established secondary neuropathology. Of note, unlike with the ERT approach, chronic lentivirus-mediated GLB1 overexpression in the GM1 gangliosidosis patient fibroblasts caused accumulation of a prelysosomal pool of β-gal, resulting in activation of the unfolded protein response and endoplasmic reticulum stress. This outcome was unsurprising in light of our in vitro biophysical findings for rhβ-gal, which include pH-dependent and concentration-dependent stability and dynamic self-association. Collectively, our results highlight that ICV-ERT is an effective therapeutic intervention for managing GM1 gangliosidosis potentially more safely than with gene therapy approaches. [ABSTRACT FROM AUTHOR]

Protein maturation in the endoplasmic reticulum (ER) depends on a fine balance between oxidative protein folding and quality control mechanisms, which together ensure high-capacity export of properly folded proteins from the ER. Oxidative protein folding needs to be regulated to avoid hyperoxidation. The folding capacity of the ER is regulated by the unfolded protein response (UPR) and ER-associated degradation (ERAD). The UPR is triggered by unfolded protein stress and leads to up-regulation of cellular components such as chaperones and folding catalysts. These components relieve stress by increasing folding capacity and up-regulating ERAD components that remove non-native proteins. Although oxidative protein folding and the UPR/ERAD pathways each are well-understood, very little is known about any direct cross-talk between them. In this study, we carried out comprehensive in vitro activity and binding assays, indicating that the oxidative protein folding relay formed by ER oxidoreductin 1 (Ero1), and protein disulfideisomerase can be inactivated by a feedback inhibition mechanism involving unfolded proteins and folding intermediates when their levels exceed the folding capacity of the system. This mechanism allows client proteins to remain mainly in the reduced state and thereby minimizes potential futile oxidation-reduction cycles and may also enhance ERAD, which requires reduced protein substrates. Relief from excess levels of non-native proteins by increasing the levels of folding factors removed the feedback inhibition. These results reveal regulatory cross-talk between the oxidative protein folding and UPR and ERAD pathways. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that ischemia/reperfusion (I/R) impairs endoplasmic reticulum (ER)-based protein folding in the heart and thereby activates an unfolded protein response sensor and effector, activated transcription factor 6 (ATF6). ATF6 then induces mesencephalic astrocyte-derived neurotrophic factor (MANF), an ER-resident protein with no known structural homologs and unclear ER function. To determine MANF's function in the heart in vivo, here we developed a cardiomyocyte-specific MANF-knockdown mouse model. MANF knockdown increased cardiac damage after I/R, which was reversed by AAV9-mediated ectopic MANF expression. Mechanistically, MANF knockdown in cultured neonatal rat ventricular myocytes (NRVMs) impaired protein folding in the ER and cardiomyocyte viability during simulated I/R. However, this was not due to MANF-mediated protection from reactive oxygen species generated during reperfusion. Because I/R impairs oxygen-dependent ER protein disulfide formation and such impairment can be caused by reductive stress in the ER, we examined the effects of the reductive ER stressor DTT. MANF knockdown in NRVMs increased cell death from DTT-mediated reductive ER stress, but not from nonreductive ER stresses caused by thapsigargin-mediated ER Ca2 depletion or tunicamycin-mediated inhibition of ER protein glycosylation. In vitro, recombinant MANF exhibited chaperone activity that depended on its conserved cysteine residues. Moreover, in cells, MANF bound to a model ER protein exhibiting improper disulfide bond formation during reductive ER stress but did not bind to this protein during nonreductive ER stress. We conclude that MANF is an ER chaperone that enhances protein folding and myocyte viability during reductive ER stress. [ABSTRACT FROM AUTHOR]

Severe congenital neutropenia (SCN) is characterized by a near absence of neutrophils, rendering individuals with this disorder vulnerable to recurrent life-threatening infections. The majority ofSCNcases arise because of germline mutations in the gene elastase, neutrophil-expressed (ELANE) encoding the neutrophil granule serine protease neutrophil elastase. Treatment with a high dose of granulocyte colony-stimulating factor increases neutrophil production and reduces infection risk. How ELANE mutations produce SCN remains unknown. The currently proposed mechanism is that ELANE mutations promote protein misfolding, resulting in endoplasmic reticulum stress and activation of the unfolded protein response (UPR), triggering death of neutrophil precursors and resulting in neutropenia. Here we studied the ELANE mutation p.G185R, often associated with greater clinical severity (e.g. decreased responsiveness to granulocyte colony-stimulating factor and increased leukemogenesis). Using an inducible expression system, we observed that this ELANE mutation diminishes enzymatic activity and granulocytic differentiation without significantly affecting cell proliferation, cell death, or UPR induction in murine myeloblast 32D and human promyelocytic NB4 cells. Impaired differentiation was associated with decreased expression of genes encoding critical hematopoietic transcription factors (Gfi1, Cebpd, Cebpe, and Spi1), cell surface proteins (Csf3r and Gr1), and neutrophil granule proteins (Mpo and Elane). Together, these findings challenge the currently prevailing model that SCN results from mutant ELANE, which triggers endoplasmic reticulum stress, UPR, and apoptosis. [ABSTRACT FROM AUTHOR]

In the presence of galactose, lithium ions activate the unfolded protein response (UPR) by inhibiting phosphoglucomutase activity and causing the accumulation of galactose-related metabolites, including galactose-1-phosphate. These metabolites also accumulate in humans who have the disease classic galactosemia. Here, we demonstrate that Saccharomyces cerevisiae yeast strains harboring a deletion of UBX4, a gene encoding a partner of Cdc48p in the endoplasmic reticulum-associated degradation (ERAD) pathway, exhibit delayed UPR activation after lithium and galactose exposure because the deletion decreases galactose-1-phosphate levels. The delay in UPR activation did not occur in yeast strains in which key ERAD or proteasomal pathway genes had been disrupted, indicating that the ubx4Δ phenotype is ERAD-independent. We also observed that the ubx4Δ strain displays decreased oxygen consumption. The inhibition of mitochondrial respiration was sufficient to diminish galactose-1-phosphate levels and, consequently, affects UPR activation. Finally, we show that the deletion of the AMP-activated protein kinase ortholog-encoding gene SNF1 can restore the oxygen consumption rate in ubx4Δ strain, thereby reestablishing galactose metabolism, UPR activation, and cellular adaption to lithium-galactose challenge. Our results indicate a role for Ubx4p in yeast mitochondrial function and highlight that mitochondrial and endoplasmic reticulum functions are intertwined through galactose metabolism. These findings also shed new light on the mechanisms of lithium action and on the pathophysiology of galactosemia. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress and unfolded protein response are cells' survival strategies to thwart disruption of proteostasis. Tumor cells are continuously being challenged by ER stress. The prion protein, PrP, normally a glycosylphosphatidylinositol (GPI)-anchored protein exists as a pro-PrP retaining its GPI-peptide signal sequence in human pancreatic ductal cell adenocarcinoma (PDAC). Higher abundance of pro-PrP indicates poorer prognosis in PDAC patients. The reason why PDAC cells express pro-PrP is unknown. Here, we report that persistent ER stress causes conversion of GPI-anchored PrP to pro-PrP via a conserved ATF6-miRNA449c-5p-PIGV axis. Mouse neurons and AsPC-1, a PDAC cell line, express GPI-anchored PrP. However, continuous culture of these cells with the ER stress inducers thapsigargin or brefeldin A results in the conversion of a GPI-anchored PrP to pro-PrP. Such a conversion is reversible; removal of the inducers allows the cells to re-express a GPI-anchored PrP. Mechanistically, persistent ER stress increases the abundance of an active ATF6, which increases the level of miRNA449c-5p (miR449c-5p). By binding the mRNA of PIGV at its 3'-UTRs, miR449c-5p suppresses the level of PIGV, a mannosyltransferase pivotal in the synthesis of the GPI anchor. Reduction of PIGV leads to disruption of the GPI anchor assembly, causing pro-PrP accumulation and enhancing cancer cell migration and invasion. The importance of ATF6-miR449c-5p-PIGV axis is recapitulated in PDAC biopsies as the higher levels of ATF6 and miR449c-5p and lower levels of PIGV are markers of poorer outcome for patients with PDAC. Drugs targeting this axis may prevent PDAC progression., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Biological functions of the highly conserved ubiquitin-like protein 5 (UBL5) are not well understood. In Caenorhabditis elegans, UBL5 is induced under mitochondrial stress to mount the mitochondrial unfolded protein response (UPR). However, the role of UBL5 in the more prevalent endoplasmic reticulum (ER) stress-UPR in the mammalian system is unknown. In the present work, we demonstrated that UBL5 was an ER stress-responsive protein, undergoing rapid depletion in mammalian cells and livers of mice. The ER stress-induced UBL5 depletion was mediated by proteasome-dependent yet ubiquitin-independent proteolysis. Activation of the protein kinase R-like ER kinase arm of the UPR was essential and sufficient for inducing UBL5 degradation. RNA-Seq analysis of UBL5-regulated transcriptome revealed that multiple death pathways were activated in UBL5-silenced cells. In agreement with this, UBL5 knockdown induced severe apoptosis in culture and suppressed tumorigenicity of cancer cells in vivo. Furthermore, overexpression of UBL5 protected specifically against ER stress-induced apoptosis. These results identify UBL5 as a physiologically relevant survival regulator that is proteolytically depleted by the UPR-protein kinase R-like ER kinase pathway, linking ER stress to cell death., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

RNA sequencing (RNA-seq) is a powerful technique for understanding cellular state and dynamics. However, comprehensive transcriptomic characterization of multiple RNA-seq datasets is laborious without bioinformatics training and skills. To remove the barriers to sequence data analysis in the research community, we have developed "RNAseqChef" (RNA-seq data controller highlighting expression features), a web-based platform of systematic transcriptome analysis that can automatically detect, integrate, and visualize differentially expressed genes and their biological functions. To validate its versatile performance, we examined the pharmacological action of sulforaphane (SFN), a natural isothiocyanate, on various types of cells and mouse tissues using multiple datasets in vitro and in vivo. Notably, SFN treatment upregulated the ATF6-mediated unfolded protein response in the liver and the NRF2-mediated antioxidant response in the skeletal muscle of diet-induced obese mice. In contrast, the commonly downregulated pathways included collagen synthesis and circadian rhythms in the tissues tested. On the server of RNAseqChef, we simply evaluated and visualized all analyzing data and discovered the NRF2-independent action of SFN. Collectively, RNAseqChef provides an easy-to-use open resource that identifies context-dependent transcriptomic features and standardizes data assessment., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)