Unfolded Protein Response Machinery Research Articles

The unfolded protein response machinery in glioblastoma genesis, chemoresistance and as a druggable target.

The role of the unfolded protein response (UPR) has been progressively unveiled over the last decade and several studies have investigated its implication in glioblastoma (GB) development. The UPR restores cellular homeostasis by triggering the folding and clearance of accumulated misfolded proteins in the ER consecutive to endoplasmic reticulum stress. In case it is overwhelmed, it induces apoptotic cell death. Thus, holding a critical role in cell fate decisions. This article, reviews how the UPR is implicated in cell homeostasis maintenance, then surveils the evidence supporting the UPR involvement in GB genesis, progression, angiogenesis, GB stem cell biology, tumor microenvironment modulation, extracellular matrix remodeling, cell fate decision, invasiveness, and grading. Next, it concurs the evidence showing how the UPR mediates GB chemoresistance-related mechanisms. The UPR stress sensors IRE1, PERK, and ATF6 with their regulator GRP78 are upregulated in GB compared to lower grade gliomas and normal brain tissue. They are activated in response to oncogenes and are implicated at different stages of GB progression, from its genesis to chemoresistance and relapse. The UPR arms can be effectors of apoptosis as mediators or targets. Recent research has established the role of the UPR in GB pathophysiology and chemoresistance. Targeting its different sensors have shown promising in overcoming GB chomo- and radioresistance and inducing apoptosis.

Fundamental and Applicative Aspects of the Unfolded Protein Response in Yeasts.

Upon the dysfunction or functional shortage of the endoplasmic reticulum (ER), namely, ER stress, eukaryotic cells commonly provoke a protective gene expression program called the unfolded protein response (UPR). The molecular mechanism of UPR has been uncovered through frontier genetic studies using Saccharomyces cerevisiae as a model organism. Ire1 is an ER-located transmembrane protein that directly senses ER stress and is activated as an RNase. During ER stress, Ire1 promotes the splicing of HAC1 mRNA, which is then translated into a transcription factor that induces the expression of various genes, including those encoding ER-located molecular chaperones and protein modification enzymes. While this mainstream intracellular UPR signaling pathway was elucidated in the 1990s, new intriguing insights have been gained up to now. For instance, various additional factors allow UPR evocation strictly in response to ER stress. The UPR machineries in other yeasts and fungi, including pathogenic species, are another important research topic. Moreover, industrially beneficial yeast strains carrying an enforced and enlarged ER have been produced through the artificial and constitutive induction of the UPR. In this article, we review canonical and up-to-date insights concerning the yeast UPR, mainly from the viewpoint of the functions and regulation of Ire1 and HAC1.

Endoplasmic stress sensor Ire1 is involved in cytosolic/nuclear protein quality control in Pichia pastoris cells independent of HAC1.

In eukaryotic species, dysfunction of the endoplasmic reticulum (ER), namely, ER stress, provokes a cytoprotective transcription program called the unfolded protein response (UPR). The UPR is triggered by transmembrane ER-stress sensors, including Ire1, which acts as an endoribonuclease to splice and mature the mRNA encoding the transcription factor Hac1 in many fungal species. Through analyses of the methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii), we revealed a previously unknown function of Ire1. In P. pastoris cells, the IRE1 knockout mutation (ire1Δ) and HAC1 knockout mutation (hac1Δ) caused only partially overlapping gene expression changes. Protein aggregation and the heat shock response (HSR) were induced in ire1Δ cells but not in hac1Δ cells even under non-stress conditions. Moreover, Ire1 was further activated upon high-temperature culturing and conferred heat stress resistance to P. pastoris cells. Our findings cumulatively demonstrate an intriguing case in which the UPR machinery controls cytosolic protein folding status and the HSR, which is known to be activated upon the accumulation of unfolded proteins in the cytosol and/or nuclei.

The anterior gradient homologue 2 (AGR2) co-localises with the glucose-regulated protein 78 (GRP78) in cancer stem cells, and is critical for the survival and drug resistance of recurrent glioblastoma: in situ and in vitro analyses.

Glioblastomas (GBs) are characterised as one of the most aggressive primary central nervous system tumours (CNSTs). Single-cell sequencing analysis identified the presence of a highly heterogeneous population of cancer stem cells (CSCs). The proteins anterior gradient homologue 2 (AGR2) and glucose-regulated protein 78 (GRP78) are known to play critical roles in regulating unfolded protein response (UPR) machinery. The UPR machinery influences cell survival, migration, invasion and drug resistance. Hence, we investigated the role of AGR2 in drug-resistant recurrent glioblastoma cells. Immunofluorescence, biological assessments and whole exome sequencing analyses were completed under in situ and in vitro conditions. Cells were treated with CNSTs clinical/preclinical drugs taxol, cisplatin, irinotecan, MCK8866, etoposide, and temozolomide, then resistant cells were analysed for the expression of AGR2. AGR2 was repressed using single and double siRNA transfections and combined with either temozolomide or irinotecan. Genomic and biological characterisations of the AGR2-expressed Jed66_GB and Jed41_GB recurrent glioblastoma tissues and cell lines showed features consistent with glioblastoma. Immunofluorescence data indicated that AGR2 co-localised with the UPR marker GRP78 in both the tissue and their corresponding primary cell lines. AGR2 and GRP78 were highly expressed in glioblastoma CSCs. Following treatment with the aforementioned drugs, all drug-surviving cells showed high expression of AGR2. Prolonged siRNA repression of a particular region in AGR2 exon 2 reduced AGR2 protein expression and led to lower cell densities in both cell lines. Co-treatments using AGR2 exon 2B siRNA in conjunction with temozolomide or irinotecan had partially synergistic effects. The slight reduction of AGR2 expression increased nuclear Caspase-3 activation in both cell lines and caused multinucleation in the Jed66_GB cell line. AGR2 is highly expressed in UPR-active CSCs and drug-resistant GB cells, and its repression leads to apoptosis, via multiple pathways.

The Role of BiP and the IRE1α-XBP1 Axis in Rhabdomyosarcoma Pathology.

Simple SummaryThe expression of BiP (GRP78), spliced XBP1 (sXBP1, nuclear XBP1), and IRE1α, were significantly associated with Rhabdomyosarcoma (RMS) in the four main RMS subtypes: alveolar (ARMS), embryonal (ERMS), pleomorphic (PRMS) and sclerosing/spindle cell (SRMS) RMS; (n = 192) compared to normal skeletal muscle tissues (n = 16). There was a significant correlation between BiP expression and the lymph node score, and between IRE1α, cytosolic XBP1 and sXBP1 expression and the stage score in all of the types of RMS. BiP and sXBP1 expression were significantly associated with all of the subtypes of RMS, whereas IRE1α was associated with ARMS, PRMS and ERMS, and cytosolic XBP1 expression was associated with ARMS and SRMS. There were correlations between BiP expression and the lymph node score in ARMS, and sXBP1 expression and the tumor score in PRMS.Background: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children, and is associated with a poor prognosis in patients presenting with recurrent or metastatic disease. The unfolded protein response (UPR) plays pivotal roles in tumor development and resistance to therapy, including RMS. Methods: In this study, we used immunohistochemistry and a tissue microarray (TMA) on human RMS and normal skeletal muscle to evaluate the expression of key UPR proteins (GRP78/BiP, IRE1α and cytosolic/nuclear XBP1 (spliced XBP1-sXBP1)) in the four main RMS subtypes: alveolar (ARMS), embryonal (ERMS), pleomorphic (PRMS) and sclerosing/spindle cell (SRMS) RMS. We also investigated the correlation of these proteins with the risk of RMS and several clinicopathological indices, such as lymph node involvement, distant metastasis, tumor stage and tumor scores. Results: Our results revealed that the expression of BiP, sXBP1, and IRE1α, but not cytosolic XBP1, are significantly associated with RMS (BiP and sXBP1 p-value = 0.0001, IRE1 p-value = 0.001) in all of the studied types of RMS tumors (n = 192) compared to normal skeletal muscle tissues (n = 16). In addition, significant correlations of BiP with the lymph node score (p = 0.05), and of IRE1α (p value = 0.004), cytosolic XBP1 (p = 0.001) and sXBP1 (p value = 0.001) with the stage score were observed. At the subtype level, BiP and sXBP1 expression were significantly associated with all subtypes of RMS, whereas IRE1α was associated with ARMS, PRMS and ERMS, and cytosolic XBP1 expression was associated with ARMS and SRMS. Importantly, the expression levels of IRE1α and sXBP1 were more pronounced in ARMS than in any of the other subtypes. The results also showed correlations of BiP with the lymph node score in ARMS (p value = 0.05), and of sXBP1 with the tumor score in PRMS (p value = 0.002). Conclusions: In summary, this study demonstrates that the overall UPR is upregulated and, more specifically, that the IRE1/sXBP1 axis is active in RMS. The subtype and stage-specific dependency on the UPR machinery in RMS may open new avenues for the development of novel targeted therapeutic strategies and the identification of specific tumor markers in this rare but deadly childhood and young-adult disease.

Unfolded Protein Response Regulates Pulmonary P53 Expression

INTRODUCTIONLung endothelial barrier dysfunction leads to severe pathologies, including the lethal Acute Respiratory Distress Syndrome. P53 has been associated with anti‐inflammatory activities. The current study employs a variety of Unfolded Protein Response (UPR) activators and inhibitors to investigate the regulation of P53 by UPR in lung cells. The outcomes of our investigation reveal a positive regulation between UPR and P53. Since it has been shown that a mild induction of UPR opposes inflammation, we suggest that P53 is involved in those protective activities in the lung.METHODSBovine Pulmonary Arterial Endothelial Cells (BPAEC) were maintained at 37°C in a humidified atmosphere of 5% CO2 – 21% O2, and were cultured in DMEM medium supplemented with 10% FBS and 1X penicillin/streptomycin. Protein levels were measured by Western Blot analysis, and were quantified using Image J software. All antibodies were purchased from Cell Signaling (Danvers, MA), and the chemicals from VWR (Radnor, PA).BPAECs were purchased from Genlantis (San Diego, CA).RESULTSBPAEC exposed to the UPR inductors Brefeldin (2 μg/ml), Dithiothreitol (1mM), or Thapsigargin (1μM) for 4, 8 and 24 hours exerted increased expression of the UPR marker Binding Immunoglobulin Protein (BiP); as well as P53. On the other hand, treatment of those cells with the UPR suppressors N‐acetyl Cysteine (1mM), Kifunensine (5μM), or the ATP‐competitive IRE1α kinase inhibiting RNase attenuator (1μM) for the same periods (4, 8, 24 hours) exerted the opposite effects. Thus, those suppressors reduced both BiP and P53 abundance in the intracellular niche.CONCLUSIONSOur study suggests that UPR is directly associated with P53 expression, and that UPR induction induces P53. Since this protein supports the endothelium function, future studies may delineate the exact components of the UPR machinery in charge of that regulation, and reveal whether those effects are associated with lung endothelium integrity.Support or Funding InformationThis work was supported by 1) R&D, Research Competitiveness Subprogram of the Louisiana Board of Regents through the Board of Regents Support Fund (LEQSF(2019‐22)‐RD‐A‐26) II) NIGMS/NIH (5P20GM103424‐15, 3P20GM103424‐15S1).

Reprogramming the unfolded protein response for replication by porcine reproductive and respiratory syndrome virus.

The unfolded protein response (UPR) in the endoplasmic reticulum (ER) constitutes a critical component of host innate immunity against microbial infections. In this report, we show that porcine reproductive and respiratory syndrome virus (PRRSV) utilizes the UPR machinery for its own benefit. We provide evidence that the virus targets the UPR central regulator GRP78 for proteasomal degradation via a mechanism that requires viral glycoprotein GP2a, while both IRE1-XBP1s and PERK-eIF2α-ATF4 signaling branches of the UPR are turned on at early stage of infection. The activated effector XBP1s was found to enter the nucleus, but ATF4 was unexpectedly diverted to cytoplasmic viral replication complexes by means of nonstructural proteins nsp2/3 to promote viral RNA synthesis. RNAi knockdown of either ATF4 or XBP1s dramatically attenuated virus titers, while overexpression caused increases. These observations reveal attractive host targets (e.g., ATF4 and XBP1s) for antiviral drugs and have implications in vaccine development.

Downregulation of the unfolded protein response is a potential mechanism for therapeutic resistance in multiple myeloma

Normal and malignant plasma cells rely on an adapted unfolded protein response (UPR) in order to withstand the endoplasmic reticulum stress associated with high immunoglobulin production. Proteasome inhibitors (PIs) overwhelm the UPR machinery, shifting from a pro-survival response to a pro-apoptotic one. Leung-Hagesteijn et al. (2013) demonstrated in multiple myeloma (MM) cell lines that downregulation of IRE1 (a principal stress sensor in the UPR and the enzyme responsible for splicing the transcription factor XBP1) leads to PI resistance.

The Unfolded Protein Response and Membrane Contact Sites: Tethering as a Matter of Life and Death?

The endoplasmic reticulum (ER) is the most extensive organelle of the eukaryotic cell and constitutes the major site of protein and lipid synthesis and regulation of intracellular Ca2+levels. To exert these functions properly, the ER network is shaped in structurally and functionally distinct domains that dynamically remodel in response to intrinsic and extrinsic cues. Moreover, the ER establishes a tight communication with virtually all organelles of the cell through specific subdomains called membrane contact sites. These contact sites allow preferential, nonvesicular channeling of key biological mediators including lipids and Ca2+between organelles and are harnessed by the ER to interface with and coregulate a variety of organellar functions that are vital to maintain homeostasis. When ER homeostasis is lost, a condition that triggers the activation of an evolutionarily conserved pathway called the unfolded protein response (UPR), the ER undergoes rapid remodeling. These dynamic changes in ER morphology are functionally coupled to the modulation or formation of contact sites with key organelles, such as mitochondria and the plasma membrane, which critically regulate cell fate decisions of the ER-stressed cells. Certain components of the UPR have been shown to facilitate the formation of contact sites through various mechanisms including remodeling of the actin cytoskeleton. In this review, we discuss old and emerging evidence linking the UPR machinery to contact site formation in mammalian cells and discuss their important role in cellular homeostasis.

In Brachypodium a complex signaling is actuated to protect cells from proteotoxic stress and facilitate seed filling.

A conserved UPR machinery is required for Brachypodium ER stress resistance and grain filling. Human and livestock diets depend on the accumulation of cereal storage proteins and carbohydrates, including mixed-linkage glucan (MLG), in the endosperm during seed development. Storage proteins and proteins responsible for the production of carbohydrates are synthesized in the endoplasmic reticulum (ER). Unfavorable conditions during growth that hamper the ER biosynthetic capacity, such as heat, can cause a potentially lethal condition known as ER stress, which activates the unfolded protein response (UPR), a signaling response designed to mitigate ER stress. The UPR relies primarily on a conserved ER-associated kinase and ribonuclease, IRE1, which splices the mRNA of a transcription factor (TF), such as bZIP60 in plants, to produce an active TF that controls the expression of ER resident chaperones. Here, we investigated activation of the UPR in Brachypodium, as a model to study the UPR in seeds of a monocotyledon species, as well as the consequences of heat stress on MLG deposition in seeds. We identified a Brachypodium bZIP60 orthologue and determined a positive correlation between bZIP60 splicing and ER stress induced by chemicals and heat. Each stress condition led to transcriptional modulation of several BiP genes, supporting the existence of condition-specific BiP regulation. Finally, we found that the UPR is elevated at the early stage of seed development and that MLG production is negatively affected by heat stress via modulation of MLG synthase accumulation. We propose that successful accomplishment of seed filling is strongly correlated with the ability of the plant to sustain ER stress via the UPR.

Novel prosurvival function of Yip1A in human cervical cancer cells: constitutive activation of the IRE1 and PERK pathways of the unfolded protein response

Cancer cells are under chronic endoplasmic reticulum (ER) stress due to hypoxia, low levels of nutrients, and a high metabolic demand for proliferation. To survive, they constitutively activate the unfolded protein response (UPR). The inositol-requiring protein 1 (IRE1) and protein kinase RNA-like ER kinase (PERK) signaling branches of the UPR have been shown to have cytoprotective roles in cancer cells. UPR-induced autophagy is another prosurvival strategy of cancer cells, possibly to remove misfolded proteins and supply nutrients. However, the mechanisms by which cancer cells exploit the UPR and autophagy machinery to promote survival and the molecules that are essential for these processes remain to be elucidated. Recently, a multipass membrane protein, Yip1A, was shown to function in the activation of IRE1 and in UPR-induced autophagy. In the present study, we explored the possible role of Yip1A in activation of the UPR by cancer cells for their survival, and found that depletion of Yip1A by RNA interference (RNAi) induced apoptotic cell death in HeLa and CaSki cervical cancer cells. Intriguingly, Yip1A was found to activate the IRE1 and PERK pathways of the UPR constitutively in HeLa and CaSki cells. Yip1A mediated the phosphorylation of IRE1 and also engaged in the transcription of PERK. The activation of these signaling pathways upregulated the expression of anti-apoptotic proteins and autophagy-related proteins. These events might enhance resistance to apoptosis and promote cytoprotective autophagy in HeLa and CaSki cells. The present study is the first to uncover a key prosurvival modulator, Yip1A, which coordinates IRE1 signaling with PERK signaling to support the survival of HeLa and CaSki cervical cancer cells.

Driving Cancer Tumorigenesis and Metastasis Through UPR Signaling.

In the tumor microenvironment, cancer cells encounter both external and internal factors that can lead to the accumulation of improperly folded proteins in the Endoplasmic Reticulum (ER) lumen, thus causing ER stress. When this happens, an adaptive mechanism named the Unfolded Protein Response (UPR) is triggered to help the cell cope with this change and restore protein homeostasis in the ER. Sequentially, one would expect that the activation of the three UPR branches, driven namely by IRE1, PERK, and ATF6, are crucial for the adaptation of cancer cells to the changing environment and thus for their survival and further propagation. Indeed, in the last few years, an increasing amount of studies has shown the implication of UPR signaling in different aspects of carcinogenesis and tumor progression. Features such as sustaining proliferation and resistance to cell death, genomic instability, altered metabolism, increased inflammation and tumor-immune infiltration, invasion and metastasis, and angiogenesis, defined as "the hallmarks of cancer", can be regulated by the UPR machinery. At the same time, new potential therapeutic interventions applicable to different kinds of cancers are being revealed. In order to describe the emerging role of UPR in cancer biology, these are the points that will be discussed in this chapter.

Abstract 436: Mfn2 is a Nodal Point Regulatory Step That Coordinates Angiogenic Phenotype in the Vascular Endothelium

Recent published data suggests that endothelial metabolism regulates angiogenesis. The key regulators of mitochondrial dynamics, mitofusins (Mfn1 and Mfn2) have been linked to cellular metabolic profile in tissues with high energetic requirements but their role in endothelial cells, were mitochondria function as signaling organelles is unknown. Here, we found that Mfn2 modulates postnatal neovascularization. Endothelial deletion of Mfn2 (Mfn2 ecko ) impaired, whereas, endothelial Mfn1 deletion (Mfn1 ecko ) did not affect clinical recovery, limb blood flow reperfusion, and capillary density compared with wild-type mice. These effects were associated with reduced eNOS activation in the ischemic leg of Mfn2 ecko mice compared with wild-type mice. Transplantation of bone marrow cells from WT to Mfn2ecko mice did not significantly improve blood perfusion in the ischemic limb of Mfn2 ecko mice. In agreement with these results, salient features of angiogenesis such as eNOS phosphorylation, cell migration, proliferation and capillary sprout formation were significantly inhibited in Mfn2 null primary mouse lung endothelial cells (Mfn2 -/- MLECs), whereas ablation of Mfn1 did not affect angiogenic signaling following VEGF stimulation. Because Mfn2 modulates the unfolded protein response (UPR) and the UPR machinery has been shown to generate angiomodulatory signals, we ascertained the effects of Mfn2 loss on UPR-mediated VEGF angiogenic signal transduction as potential mechanism. Mfn2 ablation sensitized Mfn2 -/- MLECs to proapototic UPR activation following VEGF treatment according to CHOP levels. Augmented CHOP levels corresponded with reduced eNOS phosphorylation. Inhibition of the UPR PERK signaling axis with PERK siRNA or reconstitution of Mfn2 (AdMfn2) reduced CHOP levels and significantly improved eNOS activation, EC proliferation and migration in Mfn2 -/- MLECs. Moreover, PERK siRNA or reconstitution of Mfn2 rescued the defect in capillary sprout formation in Mfn2 ecko mice. These data suggest that Mfn2 regulate angiogenic signaling in a UPR PERK axis-dependent manner and extend the role of Mfn2 beyond mitochondrial dynamics, metabolism and ER stress to angiogenesis and reparative vascular remodeling.

Unfolded Protein Response Pathways in Bloodstream-Form Trypanosoma brucei?

The unfolded protein response (UPR) is a stress mechanism to cope with misfolded proteins in the early secretory pathway, the hallmark being transcriptional upregulation of endoplasmic reticulum (ER) molecular chaperones such as BiP and protein disulfide isomerase. Despite the lack of transcriptional regulation and the absence of the classical UPR machinery, African trypanosomes apparently respond to persistent ER stress by a UPR-like response, including upregulation of BiP, and a related spliced leader silencing (SLS) response whereby SL RNA transcription is shut down. Initially observed by knockdown of the secretory protein translocation machinery, both responses are also induced by chemical agents known to elicit UPR in mammalian cells (H. Goldshmidt, D. Matas, A. Kabi, A. Carmi, R. Hope, S. Michaeli, PLoS Pathog 6:e1000731, 2010, http://dx.doi.org/10.1371/journal.ppat.1000731). As these findings were generated primarily in procyclic-stage trypanosomes, we have investigated both responses in pathogenic bloodstream-stage parasites. RNA interference (RNAi) silencing of the core translocon subunit Trypanosoma brucei Sec61α (TbSec61α) failed to induce either response. Interestingly, cell growth halted within 16 h of silencing, but sufficient TbSec61α remained to allow full competence for translocation of nascent secretory proteins for up to 24 h, indicating that replication is finely coupled with the capacity to synthesize and transport secretory cargo. Tunicamycin and thapsigargin at concentrations compatible with short-term (4 h) and long-term (24 h) viability also failed to induce any of the indicators of UPR-like or SLS responses. Dithiothreitol (DTT) was lethal at all concentrations tested. These results indicate that UPR-like and SLS responses to persistent ER stress do not occur in bloodstream-stage trypanosomes.

Exogenous salicylic acid activates two signaling arms of the unfolded protein response in Arabidopsis.

The unfolded protein response (UPR) is a highly conserved cellular response that prevents abnormal maturation of proteins in the endoplasmic reticulum (ER). The expression of genes encoding ER chaperones is induced during the UPR. In the Arabidopsis UPR, two membrane-bound transcription factors, bZIP60 and bZIP28, activate the expression of those genes. bZIP60 is regulated by unconventional cytoplasmic splicing catalyzed by inositol requiring enzyme 1 (IRE1), which is located in the ER membrane. bZIP28 is regulated by intramembrane proteolysis. Pathogen infection and salicylic acid (SA) have been reported to induce the expression of some UPR genes. Here, we show that UPR genes including BiP3, a marker gene of the Arabidopsis UPR, are induced by exogenous SA treatment and activation of bZIP60 in an IRE1-dependent manner. The induction of gene expression and activation of bZIP60 were independent of NPR1 and HsfB1 under these experimental conditions. We generated antibodies to detect the proteolytic products of bZIP28 after SA treatment. An assay using these antibodies showed that SA activated bZIP28, as well as activating bZIP60 through IRE1. Together, these results show that exogenous SA treatment activates two signaling arms of the Arabidopsis UPR. We propose a possible mechanism of activation of the UPR machinery by SA.

VEGF Signals through ATF6 and PERK to Promote Endothelial Cell Survival and Angiogenesis in the Absence of ER Stress

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) initiates IRE1α, ATF6, and PERK cascades, leading to a transcriptional/translational response known as unfolded protein response (UPR). Here we show that VEGF activates UPR mediators through a PLCγ-mediated crosstalk with the mTORC1 complex without accumulation of unfolded proteins in the ER. Activation of ATF6 and PERK contributes to the survival effect of VEGF on endothelial cells (ECs) by positively regulating mTORC2-mediated phosphorylation of AKT on Ser473, which is required for full activity of AKT. Low levels of CHOP allow ECs to evade the proapoptotic effect of this UPR product. Depletion of PLCγ, ATF6, or eIF2α dramatically inhibited VEGF-induced vascularization in mouse Matrigel plugs, suggesting that the ER and the UPR machinery constitute components of the VEGF signaling circuit that regulates EC survival and angiogenesis, extending their role beyond adaptation to ER stress.

The ER quality control and ER associated degradation machineries are vital for viral pathogenesis

The endoplasmic reticulum (ER) is central to protein production and membrane lipid synthesis. The unfolded protein response (UPR) supports cellular metabolism by ensuring protein quality control in the ER. Most positive strand RNA viruses cause extensive remodeling of membranes and require active membrane synthesis to promote infection. How viruses interact with the cellular machinery controlling membrane metabolism is largely unknown. Furthermore, there is mounting data pointing to the importance of the UPR and ER associated degradation (ERAD) machineries in viral pathogenesis in eukaryotes emerging topic. For many viruses, the UPR is an early event that is essential for persistent infection and benefits virus replication. In addition, many viruses are reported to commandeer ER resident chaperones to contribute to virus replication and intercellular movement. In particular, calreticulin, the ubiquitin machinery, and the 26S proteasome are most commonly identified components of the UPR and ERAD machinery that also regulate virus infection. In addition, researchers have noted a link between UPR and autophagy. It is well accepted that positive strand RNA viruses use autophagic membranes as scaffolds to support replication and assembly. However this topic has yet to be explored using plant viruses. The goal of research on this topic is to uncover how viruses interact with this ER-related machinery and to use this information for designing novel strategies to boost immune responses to virus infection.

Role of the unfolded protein response in β cell compensation and failure during diabetes.

Pancreatic β cell failure leads to diabetes development. During disease progression, β cells adapt their secretory capacity to compensate the elevated glycaemia and the peripheral insulin resistance. This compensatory mechanism involves a fine-tuned regulation to modulate the endoplasmic reticulum (ER) capacity and quality control to prevent unfolded proinsulin accumulation, a major protein synthetized within the β cell. These signalling pathways are collectively termed unfolded protein response (UPR). The UPR machinery is required to preserve ER homeostasis and β cell integrity. Moreover, UPR actors play a key role by regulating ER folding capacity, increasing the degradation of misfolded proteins, and limiting the mRNA translation rate. Recent genetic and biochemical studies on mouse models and human UPR sensor mutations demonstrate a clear requirement of the UPR machinery to prevent β cell failure and increase β cell mass and adaptation throughout the progression of diabetes. In this review we will highlight the specific role of UPR actors in β cell compensation and failure during diabetes.

Nrf2 and Nrf1 signaling and ER stress crosstalk: implication for proteasomal degradation and autophagy.

The endoplasmic reticulum (ER) lumen is chemically complex and crowded with polypeptides in different stages of assembly. ER quality control monitors chaperone-assisted protein folding, stochastic errors and off-pathway intermediates. In acute conditions, potentially toxic polypeptides overflow the capacity of the chaperone system and lead to ER stress. Activation of the unfolded protein response (UPR) following ER stress buys time for non-native polypeptides to refold or be eliminated; otherwise cell death occurs. The clearance routes for deleterious proteins are endoplasmic reticulum-associated degradation (ERAD) and ER stress-activated autophagy. The ERAD pathway is a chaperone and proteasome-mediated polypeptide degradation, while autophagy applies to wider range of substances. ER stress signal transduction recruits diverse molecules and pathways upon UPR induction to compensate stress condition. NF-E2-related factor 1 (Nrf1) and Nrf2 are two transcription factors mostly known by their induction through an antioxidant response; they can also be activated by UPR machinery. Discovery of diverse molecules downstream of Nrf1 and Nrf2 has expanded our understanding of the biological impacts of these transcription factors beyond classic antioxidant activation. In this review, we summarize our current understanding of mutual relationships between Nrf1, Nrf2, and ER stress clearance mechanisms and highlight the crosstalk of specific molecules mediating these correlations.

Differential unfolded protein response during Chikungunya and Sindbis virus infection: CHIKV nsP4 suppresses eIF2α phosphorylation

Chikungunya (CHIKV) and Sindbis (SINV) are arboviruses belonging to the alphavirus genus within the Togaviridae family. They cause frequent epidemics of febrile illness and long-term arthralgic sequelae that affect millions of people each year. Both viruses replicate prodigiously in infected patients and in vitro in mammalian cells, suggesting some level of control over the host cellular translational machinery that senses and appropriately directs the cell’s fate through the unfolded protein response (UPR). The mammalian UPR involves BIP (or GRP78), the master sensor in the endoplasmic reticulum (ER) together with the three downstream effector branches: inositol-requiring ser/thr protein kinase/endonuclease (IRE-1), PKR-like ER resident kinase (PERK) and activating transcription factor 6 (ATF-6). Through careful analysis of CHIKV and SINV infections in cell culture we found that the former selectively activates ATF-6 and IRE-1 branches of UPR and suppresses the PERK pathway. By separately expressing each of the CHIKV proteins as GFP-fusion proteins, we found that non-structural protein 4 (nsP4), which is a RNA-dependent-RNA polymerase, suppresses the serine-51 phosphorylation of eukaryotic translation initiation factor, alpha subunit (eIF2α), which in turn regulates the PERK pathway. This study provides insight into a mechanism by which CHIKV replication responds to overcome the host UPR machinery.