Protein Folding Capacity Of Endoplasmic Reticulum Research Articles

Understanding the Unfolded Protein Response (UPR) Pathway: Insights into Neuropsychiatric Disorders and Therapeutic Potentials.

The Unfolded Protein Response (UPR) serves as a critical cellular mechanism dedicated to maintaining protein homeostasis, primarily within the endoplasmic reticulum (ER). This pathway diligently responds to a variety of intracellular indicators of ER stress with the objective of reinstating balance by diminishing the accumulation of unfolded proteins, amplifying the ER's folding capacity, and eliminating slow-folding proteins. Prolonged ER stress and UPR irregularities have been linked to a range of neuropsychiatric disorders, including major depressive disorder, bipolar disorder, and schizophrenia. This review offers a comprehensive overview of the UPR pathway, delineating its activation mechanisms and its role in the pathophysiology of neuropsychiatric disorders. It highlights the intricate interplay within the UPR and its profound influence on brain function, synaptic perturbations, and neural developmental processes. Additionally, it explores evolving therapeutic strategies targeting the UPR within the context of these disorders, underscoring the necessity for precision and further research to effective treatments. The research findings presented in this work underscore the promising potential of UPR-focused therapeutic approaches to address the complex landscape of neuropsychiatric disorders, giving rise to optimism for improving outcomes for individuals facing these complex conditions.

Unfolded Protein Response Signaling in Liver Disorders: A 2023 Updated Review.

Endoplasmic reticulum (ER) is the site for synthesis and folding of secreted and transmembrane proteins. Disturbance in the functioning of ER leads to the accumulation of unfolded and misfolded proteins, which finally activate the unfolded protein response (UPR) signaling. The three branches of UPR-IRE1 (Inositol requiring enzyme 1), PERK (Protein kinase RNA-activated (PKR)-like ER kinase), and ATF6 (Activating transcription factor 6)-modulate the gene expression pattern through increased expression of chaperones and restore ER homeostasis by enhancing ER protein folding capacity. The liver is a central organ which performs a variety of functions which help in maintaining the overall well-being of our body. The liver plays many roles in cellular physiology, blood homeostasis, and detoxification, and is the main site at which protein synthesis occurs. Disturbance in ER homeostasis is triggered by calcium level imbalance, change in redox status, viral infection, and so on. ER dysfunction and subsequent UPR signaling participate in various hepatic disorders like metabolic (dysfunction) associated fatty liver disease, liver cancer, viral hepatitis, and cholestasis. The exact role of ER stress and UPR signaling in various liver diseases is not fully understood and needs further investigation. Targeting UPR signaling with drugs is the subject of intensive research for therapeutic use in liver diseases. The present review summarizes the role of UPR signaling in liver disorders and describes why UPR regulators are promising therapeutic targets.

Activation of the PERK branch of the unfolded protein response during production reduces specific productivity in CHO cells via downregulation of PDGFRa and IRE1a signaling.

During the course of biopharmaceutical production, heterologous protein expression in Chinese hamster ovary (CHO) cells imposes a high proteostatic burden that requires cellular adaptation. To mitigate such burden, cells utilize the unfolded protein response (UPR), which increases endoplasmic reticulum (ER) capacity to accommodate elevated rates of protein synthesis and folding. In this study, we show that during production the UPR regulates growth factor signaling to modulate growth and protein synthesis. Specifically, the protein kinase R-like ER kinase (PERK) branch of the UPR is responsible for transcriptional down-regulation of platelet-derived growth factor receptor alpha (PDGFRa) and attenuation of the IRE1-alpha (IRE1a) branch of the UPR. PERK knockout (KO) cell lines displayed reduced growth and viability due to higher rates of apoptosis despite having stabilized PDGFRa levels. Knocking out PERK in an apoptosis impaired (Bax/Bak double KO) antibody-expressing cell line prevented apoptotic cell death and revealed that apoptosis was likely triggered by increased ER stress and reactive oxygen species levels in the PERK KO hosts. Our findings suggest that attenuation of IRE1a and PDGFRa signaling by the PERK branch of the UPR reduces ER protein folding capacity and hence specific productivity of CHO cells in order to mitigate UPR and prevent apoptotic cell death. Last, Bax/Bak/PERK triple KO CHO cell lines displayed 2-3 folds higher specific productivity and titer (up to 8 g/L), suggesting that modulation of PERK signaling during production processes can greatly improve specific productivity in CHO cells.

Liraglutide Counteracts Endoplasmic Reticulum Stress in Palmitate-Treated Hypothalamic Neurons without Restoring Mitochondrial Homeostasis

One feature of high-fat diet-induced neurodegeneration in the hypothalamus is an increased level of palmitate, which is associated with endoplasmic reticulum (ER) stress, loss of CoxIV, mitochondrial fragmentation, and decreased abundance of MC4R. To determine whether antidiabetic drugs protect against ER and/or mitochondrial dysfunction by lipid stress, hypothalamic neurons derived from pre-adult mice and neuronal Neuro2A cells were exposed to elevated palmitate. In the hypothalamic neurons, palmitate exposure increased expression of ER resident proteins, including that of SERCA2, indicating ER stress. Liraglutide reverted such altered ER proteostasis, while metformin only normalized SERCA2 expression. In Neuro2A cells liraglutide, but not metformin, also blunted dilation of the ER induced by palmitate treatment, and enhanced abundance and expression of MC4R at the cell surface. Thus, liraglutide counteracts, more effectively than metformin, altered ER proteostasis, morphology, and folding capacity in neurons exposed to fat. In palmitate-treated hypothalamic neurons, mitochondrial fragmentation took place together with loss of CoxIV and decreased mitochondrial membrane potential (MMP). Metformin, but not liraglutide, reverted mitochondrial fragmentation, and both liraglutide and metformin did not protect against either loss of CoxIV abundance or MMP. Thus, ER recovery from lipid stress can take place in hypothalamic neurons in the absence of recovered mitochondrial homeostasis.

Unfolded protein response in balancing plant growth and stress tolerance.

The ER (endoplasmic reticulum) is the largest membrane-bound multifunctional organelle in eukaryotic cells, serving particularly important in protein synthesis, modification, folding and transport. UPR (unfolded protein response) is one of the systematized strategies that eukaryotic cells employ for responding to ER stress, a condition represents the processing capability of ER is overwhelmed and stressed. UPR is usually triggered when the protein folding capacity of ER is overloaded, and indeed, mounting studies were focused on the stress responding side of UPR. In plants, beyond stress response, accumulating evidence suggests that UPR is essential for growth and development, and more importantly, the necessity of UPR in this regard requires its endogenous basal activation even without stress. Then plants must have to fine tune the activation level of UPR pathway for balancing growth and stress response. In this review, we summarized the recent progresses in plant UPR, centering on its role in controlling plant reproduction and root growth, and lay out some outstanding questions to be addressed in the future.

Rescue of α-synuclein aggregation in Parkinson’s patient neurons by synergistic enhancement of ER proteostasis and protein trafficking

Neurodegenerative disorders are characterized by a collapse in proteostasis, as shown by the accumulation of insoluble protein aggregates in the brain. Proteostasis involves a balance of protein synthesis, folding, trafficking, and degradation, but how aggregates perturb these pathways is unknown. Using Parkinson's disease (PD) patient midbrain cultures, we find that aggregated α-synuclein induces endoplasmic reticulum (ER) fragmentation and compromises ER protein folding capacity, leading to misfolding and aggregation of immature lysosomal β-glucocerebrosidase. Despite this, PD neurons fail to initiate the unfolded protein response, indicating perturbations in sensing or transducing protein misfolding signals in the ER. Small molecule enhancement of ER proteostasis machinery promotes β-glucocerebrosidase solubility, while simultaneous enhancement of trafficking improves ER morphology, lysosomal function, and reduces α-synuclein. Our studies suggest that aggregated α-synuclein perturbs the ability of neurons to respond to misfolded proteins in the ER, and that synergistic enhancement of multiple proteostasis branches may provide therapeutic benefit in PD.

Porcine epidemic diarrhea virus infection induces endoplasmic reticulum stress and unfolded protein response in jejunal epithelial cells of weaned pigs.

Porcine epidemic diarrhea virus (PEDV) infection leads to diarrhea and subsequently to decreased feed efficiency and growth in weaned pigs. Given that few studies have addressed the host-virus interaction in vivo, this study focused on endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in jejunal epithelial cells during PEDV infection. Eight-week-old pigs (n = 64) were orally inoculated with PEDV IN19338 strain (n = 40) or sham-inoculated (n = 24) and analyzed for PEDV viral RNA shedding using reverse transcription-quantitative polymerase chain reaction and for viral antigen within enterocytes using immunohistochemistry (IHC). ER stress was analyzed in a subset of 9 PEDV-inoculated pigs with diarrhea, detectable viral RNA, and viral antigen (PEDV-immunopositive pigs). Compared with control pigs, PEDV-immunopositive pigs had a reduced ratio of villus height to crypt depth in the jejunum (P = .002, n = 9 per group), consistent with intestinal injury. The protein levels of ATF6, IRE1, PERK, XBP1u, ATF4, GRP78, and caspase-3 were assessed in jejunal epithelial cells at the villus tips via IHC. Both ER stress and UPR were demonstrated in PEDV-immunopositive pigs by the increased expression of ATF6 (P = .047), IRE1 (P = .007), and ATF4 (P = .001). The expression of GRP78 (P = .024) and caspase-3 (P = .004) were also increased, indicating an accompanying increase in ER protein folding capacity and apoptosis. Overall, these results reveal that PEDV infection induces ER stress and UPR in intestinal epithelial cells of weaned pigs.

Living Dangerously: Protective and Harmful ER Stress Responses in Pancreatic β-Cells.

Type 2 diabetes (T2D) is a growing cause of poor health, psychosocial burden, and economic costs worldwide. The pancreatic β-cell is a cornerstone of metabolic physiology. Insulin deficiency leads to hyperglycemia, which was fatal before the availability of therapeutic insulins; even partial deficiency of insulin leads to diabetes in the context of insulin resistance. Comprising only an estimated 1 g or <1/500th of a percent of the human body mass, pancreatic β-cells of the islets of Langerhans are a vulnerable link in metabolism. Proinsulin production constitutes a major load on β-cell endoplasmic reticulum (ER), and decompensated ER stress is a cause of β-cell failure and loss in both type 1 diabetes (T1D) and T2D. The unfolded protein response (UPR), the principal ER stress response system, is critical for maintenance of β-cell health. Successful UPR guides expansion of ER protein folding capacity and increased β-cell number through survival pathways and cell replication. However, in some cases the ER stress response can cause collateral β-cell damage and may even contribute to diabetes pathogenesis. Here we review the known beneficial and harmful effects of UPR pathways in pancreatic β-cells. Improved understanding of this stress response tipping point may lead to approaches to maintain β-cell health and function.

Boosting endoplasmic reticulum folding capacity reduces unfolded protein response activation and intracellular accumulation of human kidney anion exchanger 1 in Saccharomyces cerevisiae.

Human kidney anion exchanger 1 (kAE1) facilitates simultaneous efflux of bicarbonate and absorption of chloride at the basolateral membrane of α-intercalated cells. In these cells, kAE1 contributes to systemic acid-base balance along with the proton pump v-H+ -ATPase and the cytosolic carbonic anhydrase II. Recent electron microscopy analyses in yeast demonstrate that heterologous expression of several kAE1 variants causes a massive accumulation of the anion transporter in intracellular membrane structures. Here, we examined the origin of these kAE1 aggregations in more detail. Using various biochemical techniques and advanced light and electron microscopy, we showed that accumulation of kAE1 mainly occurs in endoplasmic reticulum (ER) membranes which eventually leads to strong unfolded protein response (UPR) activation and severe growth defect in kAE1 expressing yeast cells. Furthermore, our data indicate that UPR activation is dose dependent and uncoupled from the bicarbonate transport activity. By using truncated kAE1 variants, we identified the C-terminal region of kAE1 as crucial factor for the increased ER stress level. Finally, a redistribution of ER-localized kAE1 to the cell periphery was achieved by boosting the ER folding capacity. Our findings not only demonstrate a promising strategy for preventing intracellular kAE1 accumulation and improving kAE1 plasma membrane targeting but also highlight the versatility of yeast as model to investigate kAE1-related research questions including the analysis of structural features, protein degradation and trafficking. Furthermore, our approach might be a promising strategy for future analyses to further optimize the cell surface targeting of other disease-related PM proteins, not only in yeast but also in mammalian cells.

USP22 promotes HER2-driven mammary carcinoma aggressiveness by suppressing the unfolded protein response

The Ubiquitin-Specific Protease 22 (USP22) is a deubiquitinating subunit of the mammalian SAGA transcriptional co-activating complex. USP22 was identified as a member of the so-called “death-from-cancer” signature predicting therapy failure in cancer patients. However, the importance and functional role of USP22 in different types and subtypes of cancer remain largely unknown. In the present study, we leveraged human cell lines and genetic mouse models to investigate the role of USP22 in HER2-driven breast cancer (HER2+-BC) and demonstrate for the first time that USP22 is required for the tumorigenic properties in murine and human HER2+-BC models. To get insight into the underlying mechanisms, we performed transcriptome-wide gene expression analyses and identified the Unfolded Protein Response (UPR) as a pathway deregulated upon USP22 loss. The UPR is normally induced upon extrinsic or intrinsic stresses that can promote cell survival and recovery if shortly activated or programmed cell death if activated for an extended period. Strikingly, we found that USP22 actively suppresses UPR induction in HER2+-BC cells by stabilizing the major endoplasmic reticulum (ER) chaperone HSPA5. Consistently, loss of USP22 renders tumor cells more sensitive to apoptosis and significantly increases the efficiency of therapies targeting the ER folding capacity. Together, our data suggest that therapeutic strategies targeting USP22 activity may sensitize tumor cells to UPR induction and could provide a novel, effective approach to treat HER2+-BC.

Defects in Protein Folding and/or Quality Control Cause Protein Aggregation in the EndoplasmicReticulum.

Protein aggregation is now a common hallmark of numerous human diseases, most of which involve cytosolic aggregates including Aβ (AD) and ⍺-synuclein (PD) in Alzheimer's disease and Parkinson's disease. However, it is also evident that protein aggregation can also occur in the lumen of the endoplasmic reticulum (ER) that leads to specific diseases due to loss of protein function or detrimental effects on the host cell, the former is inherited in a recessive manner where the latter are dominantly inherited. However, the mechanisms of protein aggregation, disaggregation and degradation in the ER are not well understood. Here we provide an overview of factors that cause protein aggregation in the ER and how the ER handles aggregated proteins. Protein aggregation in the ER can result from intrinsic properties of the protein (hydrophobic residues in the ER), oxidative stress or nutrient depletion. The ER has quality control mechanisms [chaperone functions, ER-associated protein degradation (ERAD) and autophagy] to ensure only correctly folded proteins exit the ER and enter the cis-Golgi compartment. Perturbation of protein folding in the ER activates the unfolded protein response (UPR) that evolved to increase ER protein folding capacity and efficiency and degrade misfolded proteins. Accumulation of misfolded proteins in the ER to a level that exceeds the ER-chaperone folding capacity is a major factor that exacerbates protein aggregation. The most significant ER resident protein that prevents protein aggregation in the ER is the heat shock protein 70 (HSP70) homologue, BiP/GRP78, which is a peptide-dependent ATPase that binds unfolded/misfolded proteins and releases them upon ATP binding. Since exogenous factors can also reduce protein misfolding and aggregation in the ER, such as chemical chaperones and antioxidants, these treatments have potential therapeutic benefit for ER protein aggregation-associated diseases.

FSHD / OPMD / MYOTONIC DYSTROPHY: P.240 Targeting unfolded protein response to resolve aggresomes accumulation in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is a rare late-onset genetic myopathy, affecting mainly eyelid and pharyngeal muscles leading to ptosis and dysphagia, respectively. OPMD is caused by a mutation in Poly-A Binding Protein Nuclear 1 (PABPN1) gene, that codes for a protein essential for proper mRNA biogenesis in nucleus. Accordingly, mutated PABPN1 in OPMD results in PABPN1 intranuclear aggregates in muscle that sequestrate mRNA and proteins and thus induce a wide range of deleterious consequences on muscle homeostasis (mitochondrial dysfunctions, calcium homeostasis disruption, fibrosis, force impairment, atrophy). We previously demonstrated that mouse model of OPMD presents muscle Endoplasmic Reticulum (ER) stress, i.e. an accumulation of misfolded proteins in ER lumen, and that restoring the physiological and resolutive Unfolded Protein Response (UPR) through guanabenz acetate treatment is efficient to decrease PABPN1 nuclear aggregates and improve muscle function in OPMD mouse (Malerba et al Hum Mol Genet 2019). To validate these results in human, we now analyze muscle biopsies from OPMD patients compared to healthy donors and observe a disruption of both key genes and proteins of UPR signalling in OPMD muscles. Interestingly, we find that the alteration is positively correlated with the clinical state of the muscle. Furthermore, we detect GRP78 (BiP), the most important ER chaperone, trapped in PABPN1 nuclear aggregates, suggesting a reduced folding capacity in ER. As a consequence, OPMD muscles present an accumulation of large beta-sheet protein aggregates called aggresomes. Consistently, we observe that human OPMD myoblasts are more sensible to develop aggresomes than healthy myoblasts in response to ER stress. Overall, our results unravel an impairment of ER folding and UPR as well as an aggresomes accumulation in human OPMD muscle. Thus, restoring the protein folding in ER as well as improving the aggresomes clearance could be a promising therapeutic strategy against OPMD.

ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

RNA editing of adenosine to inosine (A to I) is catalyzed by ADAR1 and dramatically alters the cellular transcriptome, although its functional roles in somatic cell reprogramming are largely unexplored. Here, we show that loss of ADAR1-mediated A-to-I editing disrupts mesenchymal-to-epithelial transition (MET) during induced pluripotent stem cell (iPSC) reprogramming and impedes acquisition of induced pluripotency. Using chemical and genetic approaches, we show that absence of ADAR1-dependent RNA editinginducesaberrant innate immune responses through the double-stranded RNA (dsRNA) sensor MDA5, unleashing endoplasmic reticulum (ER) stress and hindering epithelial fate acquisition. We found that A-to-I editing impedes MDA5 sensing and sequestration of dsRNAs encoding membrane proteins, which promote ER homeostasis by activating the PERK-dependent unfolded protein response pathway to consequently facilitate MET. This study therefore establishes a critical role for ADAR1 and its A-to-I editing activity duringcell fate transitions and delineates a key regulatory layer underlying MET to control efficient reprogramming.

Hyperthermia and protein homeostasis: Cytoprotection and cell death.

Protein homeostasis or proteostasis, the correct balance between production and degradation of proteins, is an essential pillar for proper cellular function. Among the several cellular mechanisms that disrupt homeostatic conditions in cancer cells, hyperthermia (HT) has shown promising anti-tumor effects. However, cancer cells are also capable of thermoresistance. Indeed, HT-induced protein denaturation and aggregation results in the up regulation of heat shock proteins, a group of molecular chaperones with cytoprotective and anti-apoptotic properties via stress-inducible transcription factor, heat shock factor 1(HSF1). Heat shock proteins assist in the refolding of misfolded proteins and aids in their elimination if they become irreversibly damaged by various stressors. Furthermore, HSF1 also initiates the unfolded protein response in the endoplasmic reticulum (ER) to assist in the protein folding capacity of ER and also promotes the translation of pro-survival proteins' mRNA such as activating transcription factor 4 (ATF 4). Moreover, HT associated induction of microRNAs is also involved in thermal resistance of cancer cells via up-regulation of anti-apoptotic Bcl-2 proteins and down regulation of pro-apoptotic Bax and caspase 3 activities. Another cellular protection in response to stressors is Autophagy, which is regulated by the Mammalian target of rapamycin (mTOR) protein. Kinase activity in mTOR phosphorylates HSF1 and promotes its nuclear translocation for heat shock protein synthesis. Over-expression of heat shock proteins are reported to up-regulate Beclin-1, an autophagy initiator. Moreover, HT-induced reactive oxygen species (ROS) generation is sensitized by transcription factor NF-E2 related factor 2 (Nrf2) and activates the cellular expression of antioxidants and autophagy gene. Furthermore, ROS also potentiates autophagy via activation of Beclin-1. Inhibition of thermotolerance can potentiate HT-induced apoptosis. Here, we outlined that heat stress alters cellular proteins which activates cellular homeostatic processes to promote cell survival and make cancer cells thermotolerant.

Regulating ER protein homeostasis by differentially processing mRNAs

The endoplasmic reticulum (ER) protein folding capacity is balanced with the protein folding burden to prevent accumulation of un‐ or misfolded proteins. The ER‐membrane resident kinase/RNase Ire1 maintains ER protein homeostasis. Ire1 forms oligomers upon activation and initiates two distinct mRNA processing programs. First, in both metazoans and Saccharomyces cerevisiae, Ire1 catalyzes the non‐conventional cytoplasmic mRNA splicing of XBP1 (metazoans) or HAC1 (S. cerevisiae)—thereby initiating a transcriptional response that increases the ER folding capacity. Second, in metazoans and Schizosaccharomyces pombe, Ire1 selectively degrades ER‐localized mRNAs—thereby post‐transcriptionally reducing the ER’s protein folding burden. Thus, Ire1 orthologs in S. cerevisiae and S. pombe are specialized to only one of the two functional outputs, while Ire1 in metazoans can perform both. Here, we show that the respective Ire1 orthologs have become specialized for their functional outputs by divergence of their RNase specificities. Using a novel screening method in bacteria, we found that Ire1’s RNase specificity is regulated by rewiring a salt bridge at Ire1’s dimer interface. In addition, RNA structural features separate the splicing substrates from the decay substrates. Using these insights, we de novo engineered non‐conventional mRNA splicing in S. pombe, which confers S. pombe with both Ire1 functional outputs.

Phosphorylation switches protein disulfide isomerase activity to maintain proteostasis and attenuate ER stress.

Accumulated unfolded proteins in the endoplasmic reticulum (ER) trigger the unfolded protein response (UPR) to increase ER protein folding capacity. ER proteostasis and UPR signaling need to be regulated in a precise and timely manner. Here, we identify phosphorylation of protein disulfide isomerase (PDI), one of the most abundant and critical folding catalysts in the ER, as an early event during ER stress. The secretory pathway kinase Fam20C phosphorylates Ser357 of PDI and responds rapidly to various ER stressors. Phosphorylation of Ser357 induces an open conformation of PDI and turns it from a "foldase" into a "holdase", which is critical for preventing protein misfolding in the ER. Phosphorylated PDI also binds to the lumenal domain of IRE1α, a major UPR signal transducer, and attenuates excessive IRE1α activity. Importantly, PDI-S359A knock-in mice display enhanced IRE1α activation and liver damage under acute ER stress. We conclude that the Fam20C-PDI axis constitutes a post-translational response to maintain ER proteostasis and plays a vital role in protecting against ER stress-induced cell death.

Sledgehammer to Scalpel: Broad Challenges to the Heart and Other Tissues Yield Specific Cellular Responses via Transcriptional Regulation of the ER-Stress Master Regulator ATF6α.

There are more than 2000 transcription factors in eukaryotes, many of which are subject to complex mechanisms fine-tuning their activity and their transcriptional programs to meet the vast array of conditions under which cells must adapt to thrive and survive. For example, conditions that impair protein folding in the endoplasmic reticulum (ER), sometimes called ER stress, elicit the relocation of the ER-transmembrane protein, activating transcription factor 6α (ATF6α), to the Golgi, where it is proteolytically cleaved. This generates a fragment of ATF6α that translocates to the nucleus, where it regulates numerous genes that restore ER protein-folding capacity but is degraded soon after. Thus, upon ER stress, ATF6α is converted from a stable, transmembrane protein, to a rapidly degraded, nuclear protein that is a potent transcription factor. This review focuses on the molecular mechanisms governing ATF6α location, activity, and stability, as well as the transcriptional programs ATF6α regulates, whether canonical genes that restore ER protein-folding or unexpected, non-canonical genes affecting cellular functions beyond the ER. Moreover, we will review fascinating roles for an ATF6α isoform, ATF6β, which has a similar mode of activation but, unlike ATF6α, is a long-lived, weak transcription factor that may moderate the genetic effects of ATF6α.

47 Inhibition of endoplasmic reticulum stress during invitro maturation improves the developmental competence of bovine cumulus-oocyte complexes

Endoplasmic reticulum (ER) stress, a dysfunction in protein-folding capacity of ER, is involved in many physiological responses including embryonic development. Evidence shows that the ER stress-induced unfolded protein response signaling pathway is associated with impairment of oocyte maturation and pre-implantation embryonic development; supplementation of culture medium with tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, improved the developmental process of oocytes and embryos by attenuating ER stress. However, no reports are available on the role of TUDCA in reducing ER stress during IVM of bovine oocytes. Therefore, the aim of this study was to examine the mechanism of TUDCA on reducing ER stress in maturation of bovine cumulus-oocyte complexes (COCs) and whether inhibition of ER stress during maturation can promote subsequent embryonic development. Bovine ovaries were collected from a local slaughterhouse, and after aspiration COCs were matured with/without TUDCA (50, 100, and 200 µM) for 22h at 38.5°C in a humidified atmosphere of 5% CO2. After IVM, we examined the maturation rate, reactive oxygen species, apoptosis, protein/mRNA expression levels, and subsequent embryonic development after IVF. The data were analysed using analysis of variance followed by the Tukey-Kramer multiple comparison test. As a result, the dose-dependent experiment shows that a 100μM concentration of TUDCA significantly increased the maturation rate and decreased the percentage of apoptotic cells in COCs and reactive oxygen species levels in denuded oocytes. Subsequently, the expression of ER stress inducible protein GRP78/BIP significantly decreased in COCs treated with 100 µM TUDCA compared with the control COCs. In addition, the mRNA expression of ER stress and pro-apoptotic markers (GRP78/BIP, PERK, IER1, ATF4, XBP1, CHOP, and BAX) in COCs were significantly decreased by TUDCA (100 µM) treatment, whereas it increased anti-apoptotic BCL2 expression. Moreover, we show that TUDCA (100 µM) supplementation enhances embryonic development by significantly increasing the blastocyst formation rate (43.6±1.8% vs. 49.7±1.3%) and decreasing the number of apoptotic cells (7.7±1.1% vs. 5.03±0.6%) in blastocysts. These findings suggest that existence of ER stress during maturation alters the developmental competence of bovine COCs. Therefore, for the first time, we demonstrate that application of TUDCA during IVM plays a crucial role in reducing ER stress and improves the meiotic maturation, oocyte quality, and subsequent embryonic development invitro.

KSHV activates unfolded protein response sensors but suppresses downstream transcriptional responses to support lytic replication.

Herpesviruses usurp host cell protein synthesis machinery to convert viral mRNAs into proteins, and the endoplasmic reticulum (ER) to ensure proper folding, post-translational modification and trafficking of secreted and transmembrane viral proteins. Overloading ER folding capacity activates the unfolded protein response (UPR), whereby sensor proteins ATF6, PERK and IRE1 initiate a stress-mitigating transcription program that accelerates catabolism of misfolded proteins while increasing ER folding capacity. Kaposi's sarcoma-associated herpesvirus (KSHV) can be reactivated from latency by chemical induction of ER stress, which causes accumulation of the XBP1s transcription factor that transactivates the viral RTA lytic switch gene. The presence of XBP1s-responsive elements in the RTA promoter suggests that KSHV evolved a mechanism to respond to ER stress. Here, we report that ATF6, PERK and IRE1 were activated upon reactivation from latency and required for efficient KSHV lytic replication; genetic or pharmacologic inhibition of each UPR sensor diminished virion production. Despite UPR sensor activation during KSHV lytic replication, downstream UPR transcriptional responses were restricted; 1) ATF6 was cleaved to activate the ATF6(N) transcription factor but ATF6(N)-responsive genes were not transcribed; 2) PERK phosphorylated eIF2α but ATF4 did not accumulate; 3) IRE1 caused XBP1 mRNA splicing, but XBP1s protein did not accumulate and XBP1s-responsive genes were not transcribed. Ectopic expression of the KSHV host shutoff protein SOX did not affect UPR gene expression, suggesting that alternative viral mechanisms likely mediate UPR suppression during lytic replication. Complementation of XBP1s deficiency during KSHV lytic replication inhibited virion production in a dose-dependent manner in iSLK.219 cells but not in TREx-BCBL1-RTA cells. However, genetically distinct KSHV virions harvested from these two cell lines were equally susceptible to XBP1s restriction following infection of naïve iSLK cells. This suggests that cell-intrinsic properties of BCBL1 cells may circumvent the antiviral effect of ectopic XBP1s expression. Taken together, these findings indicate that while XBP1s plays an important role in reactivation from latency, it can inhibit virus replication at a later step, which the virus overcomes by preventing its synthesis. These findings suggest that KSHV hijacks UPR sensors to promote efficient viral replication while sustaining ER stress.

Abstract P116: Inhibition of Ang II-Induced Mitochondrial Fission and Endoplasmic Reticulum Stress Attenuates Protein Aggregate Accumulation, Inflammation, and Senescence in Vascular Smooth Muscle Cells

Aging is a non-modifiable risk factor for most cardiovascular diseases (CVDs) including hypertension and abdominal aortic aneurysms (AAA). As much as 14-year difference was noted in vascular aging assessed by arterial stiffness in healthy middle age human population, suggesting that premature vascular aging is a promising therapeutic target to combat against CVD. The molecular mechanisms involved in Ang II induction of premature vascular aging and how these contribute to CVD development are poorly understood. The objective of this study was to elucidate involvement of mitochondria and endoplasmic reticulum (ER) dynamics in Ang II-induced senescence in vascular smooth muscle cells (VSMCs), and how these contribute to CVDs. Ang II is known to promote mitochondrial fission and increases oxidative stress, and subsequent ER stress. Thus, chronic activation of Ang II signaling has been reported here to cause proteotoxicity due to accumulation of protein aggregates. Sodium 4-phenylbutyrate (PBA) increases ER folding capacity to restore proper protein folding and attenuate activation of UPR. Our in vitro data show Ang II enhances protein aggregate formation and PBA pretreatment alleviates their cellular accumulation in both size and number. Treatment of rat aortic VSMCs with 4-PBA or mitochondrial fission inhibitor mdivi1 mitigates Ang II induced VSMC senescence, detected by 4% (p=0.04) and 5% (p=0.02) reduction in senescence-associated β galactosidase (SA-β gal) positive cells, respectively. Ang II induced VSMC inflammation was monitored by THP-1 adhesion assay with a 1.87 fold increase in leukocyte adhesion (p=0.017), which was attenuated by PBA. In vivo , male C57/B6 mice infused with Ang II (4 weeks) with beta-aminopropionitrile (drinking water) (AAA model) +/- mdivi1 25 mg/kg IP 3x per week), which reduced AAA development, ER stress, leukocyte infiltration, and senescence. Taken together, these data represent a unique signaling pathway whereby Ang II-induced mitochondrial fission contributes to disturbed proteostasis, culminating in VSMC senescence and vascular inflamm-aging. The elucidation of Ang II contribution to irreversible vascular senescence sheds light on potential alternative therapeutic targets for high-risk CVD populations.