Cellular Unfolded Protein Response Research Articles

Pentapeptide PYRAE triggers ER stress-mediated apoptosis of breast cancer cells in mice by targeting RHBDF1-BiP interaction.

Reinforced cellular responses to endoplasmic reticulum (ER) stress are caused by a variety of pathological conditions including cancers. Human rhomboid family-1 protein (RHBDF1), a multiple transmembrane protein located mainly on the ER, has been shown to promote cancer development, while the binding immunoglobulin protein (BiP) is a key regulator of cellular unfolded protein response (UPR) for the maintenance of ER protein homeostasis. In this study, we investigated the role of RHBDF1 in maintaining ER protein homeostasis in breast cancer cells. We showed that deleting or silencing RHBDF1 in breast cancer cell lines MCF-7 and MDA-MB-231 caused marked aggregation of unfolded proteins in proximity to the ER. We demonstrated that RHBDF1 directly interacted with BiP, and this interaction had a stabilizing effect on the BiP protein. Based on the primary structural motifs of RHBDF1 involved in BiP binding, we found a pentapeptide (PE5) targeted BiP and inhibited BiP ATPase activity. SPR assay revealed a binding affinity of PE5 toward BiP (Kd = 57.7 μM). PE5 (50, 100, 200 μM) dose-dependently promoted ER protein aggregation and ER stress-mediated cell apoptosis in MCF-7 and MDA-MB-231 cells. In mouse 4T1 breast cancer xenograft model, injection of PE5 (10 mg/kg, s.c., every 2 days for 2 weeks) significantly inhibited the tumor growth with markedly increased ER stress and apoptosis-related proteins in tumor tissues. Our results suggest that the ability of RHBDF1 to maintain BiP protein stability is critical to ER protein homeostasis in breast cancer cells, and that the pentapeptide PE5 may serve as a scaffold for the development of a new class of anti-BiP inhibitors.

Effects of Spatial Expression of Activating Transcription Factor 4 on the Pathogenicity of Two Phenotypes of Bovine Viral Diarrhea Virus by Regulating the Endoplasmic Reticulum-Mediated Autophagy Process.

The endoplasmic reticulum (ER) stress response is a highly conserved stress-defense mechanism and activates the adaptive unfolded protein response (UPR) to mitigate imbalance. The ER stress-activated signaling pathways can also trigger autophagy to facilitate cellular repair. Bovine viral diarrhea virus (BVDV) utilizes the host cellular ER as the primary site of the life cycle. However, the interplay between cellular ER stress and BVDV replication remains unclear. This report reveals that cytopathic (cp) and noncytopathic (ncp) BVDV have distinct strategies to regulate UPR mechanisms and ER stress-mediated autophagy for their own benefit. Immunoblot analysis revealed that cp and ncp BVDV differentially regulated the abundance of ER chaperone GRP78 for viral replication, while the protein kinase RNA-like ER kinase (PERK)-eukaryotic translation initiation factor 2 subunit α (eIF2α)-activating transcription factor 4 (ATF4) pathway of the UPR was switched on at different stages of infection. Pretreatment with ER stress inducer promoted virion replication, but RNA interference (RNAi) knockdown of ATF4 in BVDV-infected cells significantly attenuated BVDV infectivity titers. More importantly, the effector ATF4 activated by cp BVDV infection translocated into the nucleus to mediate autophagy, but ATF4 was retained in the cytoplasm during ncp BVDV infection. In addition, we found that cp BVDV core protein was localized in the ER to induce ER stress-mediated autophagy. Overall, the potential therapeutic target ATF4 may contribute to the global eradication campaign of BVDV. IMPORTANCE The ER-tropic viruses hijack the host cellular ER as the replication platform of the life cycle, which can lead to strong ER stress. The UPR and related transcriptional cascades triggered by ER stress play a crucial role in viral replication and pathogenesis, but little is known about these underlying mechanisms. Here, we report that cytopathic and noncytopathic BVDV use different strategies to reprogram the cellular UPR and ER stress-mediated autophagy for their own advantage. The cytopathic BVDV unconventionally downregulated the expression level of GRP78, creating perfect conditions for self-replication via the UPR, and the noncytopathic BVDV retained ATF4 in the cytoplasm to provide an advantage for its persistent infection. Our findings provide new insights into exploring how BVDV and other ER-tropic viruses reprogram the UPR signaling pathway in the host cells for replication and reveal the attractive host target ATF4 for new antiviral agents.

Role of endoplasmic reticulum stress in hepatic glucose and lipid metabolism and therapeutic strategies for metabolic liver disease.

The endoplasmic reticulum is the most abundant membrane organelle in eukaryotic cells. It is an important site of membrane and secretory protein folding and glycolipid synthesis and transport, and it is responsible for regulating intracellular calcium homeostasis. When the load of protein synthesis and folding exceeds the processing capacity of the endoplasmic reticulum, the excessive accumulation of misfolded proteins leads to endoplasmic reticulum stress and activates the cellular unfolded protein response. Hepatocytes contain an abundance of smooth and rough endoplasmic reticulum, which can sense changes in various nutritional metabolic and external stimuli and mediate the regulation of glucose and lipid metabolism by activating the unfolded protein response signaling pathways. Endoplasmic reticulum stress plays a very important role in metabolic regulation and in the occurrence and development of liver diseases. This review summarizes the recent research progress on the unfolded protein response and hepatic glucose and lipid metabolism and discusses the mechanism linking endoplasmic reticulum stress with glucose and lipid metabolism disorders and related metabolic liver diseases to improve the understanding of the molecular pathological basis of major chronic diseases such as obesity, type II diabetes and nonalcoholic fatty liver disease.

NCS1 overexpression restored mitochondrial activity and behavioral alterations in a zebrafish model of Wolfram syndrome

Wolfram syndrome (WS) is a rare neurodegenerative disease resulting in deafness, optic atrophy, diabetes, and neurological disorders. Currently, no treatment is available for patients. The mutated gene, WFS1, encodes an endoplasmic reticulum (ER) protein, Wolframin. We previously reported that Wolframin regulated the ER-mitochondria Ca2+ transfer and mitochondrial activity by protecting NCS1 from degradation in patients' fibroblasts. We relied on a zebrafish model of WS, the wfs1ab KO line, to analyze the functional and behavioral impact of NCS1 overexpression as a novel therapeutic strategy. The wfs1ab KO line showed an increased locomotion in the visual motor and touch-escape responses. The absence of wfs1 did not impair the cellular unfolded protein response, in basal or tunicamycin-induced ER stress conditions. In contrast, metabolic analysis showed an increase in mitochondrial respiration in wfs1ab KO larvae. Interestingly, overexpression of NCS1 using mRNA injection restored the alteration of mitochondrial respiration and hyperlocomotion. Taken together, these data validated the wfs1ab KO zebrafish line as a pertinent experimental model of WS and confirmed the therapeutic potential of NCS1. The wfs1ab KO line therefore appeared as an efficient model to identify novel therapeutic strategies, such as gene or pharmacological therapies targeting NCS1 that will correct or block WS symptoms.

Fat body Ire1 regulates lipid homeostasis through the Xbp1s-FoxO axis in Drosophila

SummaryThe endoplasmic reticulum (ER)-resident transmembrane protein kinase/RNase Ire1 is a conserved sensor of the cellular unfolded protein response and has been implicated in lipid homeostasis, including lipid synthesis and transport, across species. Here we report a novel catabolic role of Ire1 in regulating lipid mobilization in Drosophila. We found that Ire1 is activated by nutrient deprivation, and, importantly, fat body-specific Ire1 deficiency leads to increased lipid mobilization and sensitizes flies to starvation, whereas fat body Ire1 overexpression results in the opposite phenotypes. Genetic interaction and biochemical analyses revealed that Ire1 regulates lipid mobilization by promoting Xbp1s-associated FoxO degradation and suppressing FoxO-dependent lipolytic programs. Our results demonstrate that Ire1 is a catabolic sensor and acts through the Xbp1s-FoxO axis to hamper the lipolytic response during chronic food deprivation. These findings offer new insights into the conserved Ire1 regulation of lipid homeostasis.

Fat Body Ire1 Regulates Lipid Homeostasis Through the Xbp1s-FoxO Axis in <i>Drosophila</i>

Summary The endoplasmic reticulum (ER)-resident transmembrane protein kinase/RNase Ire1 is a conserved sensor of the cellular unfolded protein response and has been implicated in lipid homeostasis, including lipid synthesis and transport, across species. Here we report a novel catabolic role of Ire1 in regulating lipid mobilization in Drosophila. We found that Ire1 is activated by nutrient deprivation, and, importantly, fat body-specific Ire1 deficiency leads to increased lipid mobilization and sensitizes flies to starvation, whereas fat body Ire1 overexpression results in the opposite phenotypes. Genetic interaction and biochemical analyses revealed that Ire1 regulates lipid mobilization by promoting Xbp1s-associated FoxO degradation and suppressing FoxO-dependent lipolytic programs. Our results demonstrate that Ire1 is a catabolic sensor and acts through the Xbp1s-FoxO axis to hamper the lipolytic response during chronic food deprivation. These findings offer new insights into the conserved Ire1 regulation of lipid homeostasis.

DIPG-57. TRANSCRIPTOMIC AND PROTEOMIC ANALYSES OF DIPG RESPONSE TO ONC201

Diffuse Intrinsic Pontine Glioma (DIPG) is an incurable pediatric brain tumor. Current standard of care has shown no improvements in survival. Here, we report our study of ONC201, a first-in-class small molecule developed by Oncoceutics, Inc., against a panel of DIPG cells in vitro and in mouse orthotopic models. ONC201 inhibits signaling through dopamine receptor D2 (DRD2), a G protein-coupled receptor (GPCR). MTT assays revealed a delayed but more robust response to ONC201, as measured by IC50 values, in DIPGs with histone H3.3-K27M expression compared to cells expressing wildtype (WT) or K27M mutant histone H3.1. Interestingly, transcriptomic profiling identified an association of this response delay with an elevation of genes controlling the cellular unfolded protein response, lysosomal and vacuole organization, and a decline in nucleic acid biosynthetic genes. These cells were also more committed to neuronal and oligodendrocytic lineage specification. By contrast, WT-H3 DIPGs that survived ONC201 treatment were stem-like and exhibited altered expression of genes controlling cell proliferation and apoptosis induction, respectively. Single cell proteomics validated the increase in anti-apoptotic proteins in these cells. Intraperitoneal administration of ONC201 for 7-weeks in mice bearing pontine xenografts of histone H3.1-K27M mutant DIPGs, caused a complete blockade of tumor growth relative to untreated controls. However, identical treatment of animals with forebrain tumors resulted only in a partial reduction in tumor burden, suggesting that the tumor microenvironment may be involved in the differential effect. These data indicate that tumor intrinsic and extrinsic factors may contribute to the response of DIPG tumors to ONC201.

A Seven-Transmembrane Protein -TM7SF3, Resides in Nuclear Speckles and Regulates Alternative Splicing

The seven-transmembrane superfamily member 3 protein (TM7SF3) is a p53-regulated homeostatic factor that attenuates the development of cellular stress and unfolded protein response. Here we show that TM7SF3 localizes to nuclear speckles; eukaryotic nuclear bodies enriched in splicing factors. This rather unexpected location of a membranal protein enables it to form stable complexes with regulators of pre-mRNA splicing including DHX15, LARP7, HNRNPU and RBM14 that co-precipitate with TM7SF3. Indeed, TM7SF3 modulates alternative splicing of >400 genes, mainly splicing at the 3’end of introns. These effects are observed both in primary human pancreatic islets and in a number of cell lines, under basal conditions and under stress. Accordingly, silencing of TM7SF3 resulted in differential expression of 1465 genes (about 7% of the human genome); with 844 and 621 genes being up- or down-regulated, respectively. Our findings implicate TM7SF3, as a resident of nuclear speckles and suggest a role for seven-transmembrane proteins as regulators of alternative splicing.

CSFV protein NS5A activates the unfolded protein response to promote viral replication

Classical swine fever is a world organization for animal health listed disease and is caused by classical swine fever virus (CSFV). CSFV can induced unfolded protein response (UPR) and whether NS5A protein plays a role in this process remains unknown. Here, we demonstrate that CSFV induced all the three signal pathways ATF6, IRE1 and PERK of UPR. Furthermore, this phenomenon may be mediated by the NS5A protein since expression of NS5A alone can achieve the same effect. In the current study, we show that NS5A can interact with GRP78 as measured by using the CO-IP and GST pulldown assays. This interaction plays a positive role in the promotion of CSFV replication. Overexpression or knockdown of GRP78 mediated by lentivirus can enhance or decrease viral replication, respectively. Our findings provide the evidence that CSFV infection can activate the cellular UPRs, in which NS5A and GRP78 play key roles in the process.

The unfolded protein response induced by Tembusu virus infection

BackgroundTembusu virus (TMUV), classified in the genus Flavivirus, causes reduced egg production and neurological problems in poultry. Flavivirus replication depends on the host endoplasmic reticulum (ER) and induces ER stress that leads to activation of the cellular unfolded protein response (UPR), an important signalling pathway that regulates many biological functions involved in viral pathogenesis and innate immunity. However, the mechanism of TMUV-induced UPR activation remains unclear.ResultsIn this study, we systematically investigated the three UPR pathways in TMUV-infected BHK-21 cells. Our results showed that expression of glucose-related protein 78 (GRP78) and GRP94 was upregulated during the course of TMUV infection. We then demonstrated that TMUV activated the PERK pathway in the early stage of infection, resulting in upregulation of ATF4, GADD34 and CHOP, with CHOP induction leading to caspase-3 activation. We also found the IRE1 pathway to be activated, leading to splicing of X box binding protein 1 (XBP1) mRNA and enhanced expression of p58IPK. Finally, we observed increased expression of ATF6 and activity of ER stress-response elements, suggesting stimulation of the ATF6 pathway. In addition, ATF6 pathway activation correlated with the induction of downstream chaperones calnexin, calreticulin, ERp57 and PDI. UPR activity was also observed by the marked elevation in GRP78 and sXBP1 levels in TMUV-infected DF-1 cells.ConclusionsThis is the first report that TMUV infection-induced ER stress activates three branches of the UPR, and these results lay the foundation for elucidating the pathogenesis of TMUV and understanding the inherent mechanism of TMUV infection as well as the host response.

Host cellular unfolded protein response signaling regulates Campylobacter jejuni invasion.

Campylobacter jejuni is a major cause of bacterial foodborne illness in humans worldwide. Bacterial entry into a host eukaryotic cell involves the initial steps of adherence and invasion, which generally activate several cell-signaling pathways that induce the activation of innate defense systems, which leads to the release of proinflammatory cytokines and induction of apoptosis. Recent studies have reported that the unfolded protein response (UPR), a system to clear unfolded proteins from the endoplasmic reticulum (ER), also participates in the activation of cellular defense mechanisms in response to bacterial infection. However, no study has yet investigated the role of UPR in C. jejuni infection. Hence, the aim of this study was to deduce the role of UPR signaling via induction of ER stress in the process of C. jejuni infection. The results suggest that C. jejuni infection suppresses global protein translation. Also, 12 h of C. jejuni infection induced activation of the eIF2α pathway and expression of the transcription factor CHOP. Interestingly, bacterial invasion was facilitated by knockdown of UPR-associated signaling factors and treatment with the ER stress inducers, thapsigargin and tunicamycin, decreased the invasive ability of C. jejuni. An investigation into the mechanism of UPR-mediated inhibition of C. jejuni invasion showed that UPR signaling did not affect bacterial adhesion to or survival in the host cells. Further, Salmonella Enteritidis or FITC-dextran intake were not regulated by UPR signaling. These results indicated that the effect of UPR on intracellular intake was specifically found in C. jejuni infection. These findings are the first to describe the role of UPR in C. jejuni infection and revealed the participation of a new signaling pathway in C. jejuni invasion. UPR signaling is involved in defense against the early step of C. jejuni invasion and thus presents a potential therapeutic target for the treatment of C. jejuni infection.

Editing N-Glycan Site Occupancy with Small-Molecule Oligosaccharyltransferase Inhibitors

The oligosaccharyltransferase (OST) is a multisubunit enzyme complex that N-glycosylates proteins in the secretory pathway and is considered to be constitutive and unregulated. However, small-molecule OST inhibitors such as NGI-1 provide a pharmacological approach for regulating N-linked glycosylation. Herein we design cell models with knockout of each OST catalytic subunit (STT3A or STT3B) to screen the activity of NGI-1 and its analogs. We show that NGI-1 targets the function of both STT3A and STT3B and use structure-activity relationships to guide synthesis of catalytic subunit-specific inhibitors. Using this approach, pharmacophores that increase STT3B selectivity are characterized and an STT3B-specific inhibitor is identified. This inhibitor has discrete biological effects on endogenous STT3B target proteins such as COX2 but does not activate the cellular unfolded protein response. Together this work demonstrates that subsets of glycoproteins can be regulated through pharmacologic inhibition of N-linked glycosylation.

Exendin-4 protects INS-1 cells against palmitate-induced apoptosis through the IRE1α-Xbp1 signaling pathway.

The anti-apoptotic effect of the incretin analog, exendin-4 (EX-4) on pancreatic β cells is mediated via the activation of protein kinase B (Akt) signaling, and its effect is partly produced through the inhibition of endoplasmic reticulum (ER) stress. However, the molecular mechanisms that underlie the effect of EX-4 on the suppression of ER stress and the upregulation of Akt signaling are poorly understood. Inositol-requiring enzyme 1 (IRE1), a member of the ER-localized transmembrane protein family, activates its downstream transcription factor X-box binding protein 1 (XBP1) to mediate a key part of the cellular unfolded protein response in order to cope with ER stress. Using the clonal rat pancreatic β cell line INS-1, the present study produced an in vitro model of ER stress using palmitate (PA) in order to determine whether the beneficial effect of EX-4 under ER stress was regulated by the IRE1α-Xbp1 signaling pathway. The results demonstrated that the reduction in ER stress and the activation Akt by EX-4 may be associated with the upregulation of IRE1α phosphorylation and the splicing of Xbp1 mRNA, which improved PA-reduced cell viability. This effect was partially abrogated by the knockdown of IRE1α with small interfering RNA. Additionally, cellular IRE1α was phosphorylated by the protein kinase A (PKA) associated with EX-4 and the activation of IRE1α, as IRE1α phosphorylation was attenuated by the inhibition of PKA with its inhibitor. In conclusion, the data identified the IRE1α-Xbp1 signaling pathway as an essential mediator that associates EX-4 with the intracellular mechanism that inhibits ER stress and activates Akt in order to regulate β cell survival. This may provide important evidence for the use of EX-4 in treatments for type 2 diabetes.

Characterization of an A-Site Selective Protein Disulfide Isomerase A1 Inhibitor.

Protein disulfide isomerase A1 (PDIA1) is an endoplasmic reticulum (ER)-localized thiol-disulfide oxidoreductase that is an important folding catalyst for secretory pathway proteins. PDIA1 contains two active-site domains (a and a'), each containing a Cys-Gly-His-Cys (CGHC) active-site motif. The two active-site domains share 37% sequence identity and function independently to perform disulfide-bond reduction, oxidation, and isomerization. Numerous inhibitors for PDIA1 have been reported, yet the selectivity of these inhibitors toward the a and a' sites is poorly characterized. Here, we identify a potent and selective PDIA1 inhibitor, KSC-34, with 30-fold selectivity for the a site over the a' site. KSC-34 displays time-dependent inhibition of PDIA1 reductase activity in vitro with a kinact/ KI of 9.66 × 103 M-1 s-1 and is selective for PDIA1 over other members of the PDI family, and other cellular cysteine-containing proteins. We provide the first cellular characterization of an a-site selective PDIA1 inhibitor and demonstrate that KSC-34 has minimal sustained effects on the cellular unfolded protein response, indicating that a-site inhibition does not induce global protein folding-associated ER stress. KSC-34 treatment significantly decreases the rate of secretion of a destabilized, amyloidogenic antibody light chain, thereby minimizing pathogenic amyloidogenic extracellular proteins that rely on high PDIA1 activity for proper folding and secretion. Given the poor understanding of the contribution of each PDIA1 active site to the (patho)physiological functions of PDIA1, site selective inhibitors like KSC-34 provide useful tools for delineating the pathological role and therapeutic potential of PDIA1.

Multiplexed Digital mRNA Expression Analysis Profiles System-Wide Changes in mRNA Abundance and Responsiveness of UPR-Specific Gene Expression Changes During Batch Culture of Recombinant Chinese Hamster Ovary Cells.

The unfolded protein response (UPR) signaling pathway is viewed as critical for setting the effectiveness of recombinant protein expression in CHO cells. In this study, Nanostring nCounter technology is used to study expression of a group of genes associated with cellular processes linked to UPR activation under ER stress and the changing environment of a batch culture. Time course induction of ER stress, using tunicamycin (TM), shows a group of genes such as Chop, Trb3, Sqstm1, Grp78, and Herpud1 respond rapidly to TM inhibition of N-glycosylation, while others such as Atf5, Odz4, and Birc5 exhibits a delayed response. In batch culture, expression of "classical" UPR markers only increases when cells enter decline phase. In addition to providing a detailed analysis of the expression of process-relevant UPR markers during batch culture and in response to imposed chemical stress, we also highlighted six genes (Herpud1, Odz4, Sqstm1, Trb3, Syvn1, and Birc5) associated with the perception of ER stress responses in recombinant CHO cells. Herpud1 (involved in ER-associated degradation) exhibits a rapid (primary) response to stress and its relationship (and that of the other five genes) to the overall cellular UPR may identify novel targets to modulate recombinant protein production in CHO cells.

Silver nanoparticles induce SH-SY5Y cell apoptosis via endoplasmic reticulum- and mitochondrial pathways that lengthen endoplasmic reticulum-mitochondria contact sites and alter inositol-3-phosphate receptor function

Silver nanoparticles (AgNPs) have many medical and commercial applications, but their effects on human health are poorly understood. The aim of this study was to assess the effect of AgNPs on the human neuroblastoma cell line SH-SY5Y and to explore their potential mechanisms of action. We found that AgNPs decreased SH-SY5Y cell viability in a dose- and time-dependent manner. Exposure to AgNPs activated endoplasmic reticulum (ER) stress, as reflected by upregulated expression of glucose-regulated protein 78 (GRP78), phosphorylated PKR-like endoplasmic reticulum kinase (p-PERK), phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α), C/EBP homology protein (CHOP), spliced X-box binding protein-1 (XBP1), and phosphorylated inositol-requiring enzyme (p-IRE), all of which are involved in the cellular unfolded protein response. Prolonged exposure of cells to AgNPs damaged calcium (Ca2+) homeostasis, increased the length of contact sites between the ER and mitochondria, altered IP3R function by the increased levels of phosphatase and tensin homolog deleted on chromosome ten (PTEN) in the ER and enhanced mitochondrial Ca2+ uptake. Finally, Ca2+ overload and disrupted homeostasis in the mitochondria triggered apoptotic cell death. Our results suggest that caution should be exercised in the use of AgNPs in humans.

Intermittent hypoxia leads to functional reorganization of mitochondria and affects cellular bioenergetics in marine molluscs.

Fluctuations in oxygen (O2) concentrations represent a major challenge to aerobic organisms and can be extremely damaging to their mitochondria. Marine intertidal molluscs are well-adapted to frequent O2 fluctuations, yet it remains unknown how their mitochondrial functions are regulated to sustain energy metabolism and prevent cellular damage during hypoxia and reoxygenation (H/R). We used metabolic control analysis to investigate the mechanisms of mitochondrial responses to H/R stress (18 h at <0.1% O2 followed by 1 h of reoxygenation) using hypoxia-tolerant intertidal clams Mercenaria mercenaria and hypoxia-sensitive subtidal scallops Argopecten irradians as models. We also assessed H/R-induced changes in cellular energy balance, oxidative damage and unfolded protein response to determine the potential links between mitochondrial dysfunction and cellular injury. Mitochondrial responses to H/R in scallops strongly resembled those in other hypoxia-sensitive organisms. Exposure to hypoxia followed by reoxygenation led to a strong decrease in the substrate oxidation (SOX) and phosphorylation (PHOS) capacities as well as partial depolarization of mitochondria of scallops. Elevated mRNA expression of a reactive oxygen species-sensitive enzyme aconitase and Lon protease (responsible for degradation of oxidized mitochondrial proteins) during H/R stress was consistent with elevated levels of oxidative stress in mitochondria of scallops. In hypoxia-tolerant clams, mitochondrial SOX capacity was enhanced during hypoxia and continued rising during the first hour of reoxygenation. In both species, the mitochondrial PHOS capacity was suppressed during hypoxia, likely to prevent ATP wastage by the reverse action of FO,F1-ATPase. The PHOS capacity recovered after 1 h of reoxygenation in clams but not in scallops. Compared with scallops, clams showed a greater suppression of energy-consuming processes (such as protein turnover and ion transport) during hypoxia, indicated by inactivation of the translation initiation factor EIF-2α, suppression of 26S proteasome activity and a dramatic decrease in the activity of Na(+)/K(+)-ATPase. The steady-state levels of adenylates were preserved during H/R exposure and AMP-dependent protein kinase was not activated in either species, indicating that the H/R exposure did not lead to severe energy deficiency. Taken together, our findings suggest that mitochondrial reorganizations sustaining high oxidative phosphorylation flux during recovery, combined with the ability to suppress ATP-demanding cellular functions during hypoxia, may contribute to high resilience of clams to H/R stress and help maintain energy homeostasis during frequent H/R cycles in the intertidal zone.

PCSK1 Variants and Human Obesity.

PCSK1, encoding prohormone convertase 1/3 (PC1/3), was one of the first genes linked to monogenic early-onset obesity. PC1/3 is a protease involved in the biosynthetic processing of a variety of neuropeptides and prohormones in endocrine tissues. PC1/3 activity is essential for the activating cleavage of many peptide hormone precursors implicated in the regulation of food ingestion, glucose homeostasis, and energy homeostasis, for example, proopiomelanocortin, proinsulin, proglucagon, and proghrelin. A large number of genome-wide association studies in a variety of different populations have now firmly established a link between three PCSK1 polymorphisms frequent in the population and increased risk of obesity. Human subjects with PC1/3 deficiency, a rare autosomal-recessive disorder caused by the presence of loss-of-function mutations in both alleles, are obese and display a complex set of endocrinopathies. Increasing numbers of genetic diagnoses of infants with persistent diarrhea has recently led to the finding of many novel PCSK1 mutations. PCSK1-deficient infants experience severe intestinal malabsorption during the first years of life, requiring controlled nutrition; these children then become hyperphagic, with associated obesity. The biochemical characterization of novel loss-of-function PCSK1 mutations has resulted in the discovery of new pathological mechanisms affecting the cell biology of the endocrine cell beyond simple loss of enzyme activity, for example, dominant-negative effects of certain mutants on wild-type PC1/3 protein, and activation of the cellular unfolded protein response by endoplasmic reticulum-retained mutants. A better understanding of these molecular and cellular pathologies may illuminate possible treatments for the complex endocrinopathy of PCSK1 deficiency, including obesity.

Clotting Factor VIII Overexpression Shows Signs of ER Stress but Does Not Cause Toxicity upon Gene Transfer

Hemophilia A is the X-linked bleeding disorder resulting from the loss of functional clotting factor VIII (FVIII). Hemophilia A patients with severe disease (<1% residual FVIII activity) experience spontaneous bleeds into the joints and closed spaces with severe morbidity. Restoration of hemostasis is managed by repeated infusions (2-3 times per week) of plasma derived or recombinant FVIII protein. While a standard treatment is available for patients, life-long infusions of FVIII protein is very expensive, has a negative impact on the patient's quality of life, and FVIII protein products are not available worldwide. Hence, there is a need to develop a more robust and cost effective treatment for hemophilia A patients. Liver-directed gene therapy using adeno-associated virus (AAV) represents a promising approach to treat hemophilia A. However, previous studies have shown that overexpression of human FVIII protein in the context of hydrodynamic delivery of plasmid vectors induces ER stress mediated through the unfolded protein response (UPR). Because human FVIII protein is inefficiently secreted into circulation, high AAV vector doses will be required to obtain therapeutic expression levels. Therefore, we sought to determine if AAV-FVIII gene delivery also triggers cellular UPR in hepatocytes in vivo. To this end, we selected to use a codon-optimized FVIII cDNA, which has been shown by our lab to significantly increase FVIII protein expression, and a high vector dose of AAV8-ApoE-hAAT-cohF8, containing a hepatocyte-specific enhancer/promoter combination. We evaluated this vector at doses of 1 x 1011 vg (4x1012 vg/kg) and 1 x 1012 vg (4x1013 vg/kg) in hemophilia A mice on a 129/BL6 background. Intravenous administration of the highest vector dose completely restored hemostasis, which was sustained and achieved super-physiological levels in some animals. Importantly, none of these mice developed inhibitors against FVIII. Next, we administered the vector at the same 2 doses to C67BL/6 mice, which show higher hepatic transduction efficiency than other strains. Experimental controls consisted of mice, with no vector or 1x1012 vg of AAV8-ApoE-hAAT-F9, expressing human factor IX (FIX) protein. Injection of tunicamycin, a potent inducer of the ER stress response, served as a positive control for all assays. Vector-treated mice were studied 2 and 4 weeks after gene transfer (n=3-4 per group). First, we evaluated the status of key molecular chaperones, known to be the mediators of the UPR: Bip, p-PERK, and p-eIF2a. Western blotting performed on the liver lysates indicated modest up-regulation of all three markers compared to normal control, but that effect was neither dose nor gene dependent. In addition, we tested the splicing of Xbp1 mRNA by PCR assay and observed low level of the 26 bp spliced fragment, indicative of the UPR, at the high vector dose. Immunohistochemistry on liver sections from each of our experimental groups including H&E staining, Tunnel staining for apoptotic cells, and reactive oxygen species staining. None of the stains yielded evidence for liver damage even in the 1x1012 AAV8-cohF8 treated mice compared to untreated controls. There was also no elevation of liver enzyme levels in plasma samples. Analysis of plasma from vector injected mice showed systemic levels of human FVIII and FIX proteins at ~30 ng/ml and ~6300 ng/ml, respectively (these ELISA-based measurements likely underestimate FVIII levels due to interference by von Willebrand factor). Overall our results suggest that over-expression of coagulation factors in hepatocytes from AAV vectors causes a mild cell stress response that is not strong enough to cause liver toxicity, is not specific for FVIII, and does impact expression or immunogenicity. DisclosuresNo relevant conflicts of interest to declare.

Crystal structures reveal transient PERK luminal domain tetramerization in endoplasmic reticulum stress signaling

Stress caused by accumulation of misfolded proteins within the endoplasmic reticulum (ER) elicits a cellular unfolded protein response (UPR) aimed at maintaining protein-folding capacity. PERK, a key upstream component, recognizes ER stress via its luminal sensor/transducer domain, but the molecular events that lead to UPR activation remain unclear. Here, we describe the crystal structures of mammalian PERK luminal domains captured in dimeric state as well as in a novel tetrameric state. Small angle X-ray scattering analysis (SAXS) supports the existence of both crystal structures also in solution. The salient feature of the tetramer interface, a helix swapped between dimers, implies transient association. Moreover, interface mutations that disrupt tetramer formation in vitro reduce phosphorylation of PERK and its target eIF2α in cells. These results suggest that transient conversion from dimeric to tetrameric state may be a key regulatory step in UPR activation.