Cytomegalovirus Downregulates IRE1 to Repress the Unfolded Protein Response

During viral infection, a massive demand for viral glycoproteins can overwhelm the capacity of the protein folding and quality control machinery, leading to an accumulation of unfolded proteins in the endoplasmic reticulum (ER). To restore ER homeostasis, cells initiate the unfolded protein response (UPR) by activating three ER-to-nucleus signaling pathways, of which the inositol-requiring enzyme 1 (IRE1)-dependent pathway is the most conserved. To reduce ER stress, the UPR decreases protein synthesis, increases degradation of unfolded proteins, and upregulates chaperone expression to enhance protein folding. Cytomegaloviruses, as other viral pathogens, modulate the UPR to their own advantage. However, the molecular mechanisms and the viral proteins responsible for UPR modulation remained to be identified. In this study, we investigated the modulation of IRE1 signaling by murine cytomegalovirus (MCMV) and found that IRE1-mediated mRNA splicing and expression of the X-box binding protein 1 (XBP1) is repressed in infected cells. By affinity purification, we identified the viral M50 protein as an IRE1-interacting protein. M50 expression in transfected or MCMV-infected cells induced a substantial downregulation of IRE1 protein levels. The N-terminal conserved region of M50 was found to be required for interaction with and downregulation of IRE1. Moreover, UL50, the human cytomegalovirus (HCMV) homolog of M50, affected IRE1 in the same way. Thus we concluded that IRE1 downregulation represents a previously undescribed viral strategy to curb the UPR.     

IRE1α disruption causes histological abnormality of exocrine tissues, increase of blood glucose level, and decrease of serum immunoglobulin level

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress. As a cellular adaptive response to ER stress, unfolded protein response (UPR) activates molecules for the quality control of ER proteins. One enzyme that plays an important role in UPR is Inositol Requiring Enzyme-1 (IRE1), which is highly conserved from yeast to humans. In particular, mammalian IRE1α activates X-box-binding protein 1 (XBP1) by unconventional splicing of XBP1 mRNA during ER stress. From analysis of knockout mice, both IRE1α and XBP1 have been shown to be essential for development and that XBP1 is necessary for the secretory machinery of exocrine glands, plasma cell differentiation, and hepatic lipogenesis. However, the essentiality of IRE1α in specific organs and tissues remains incompletely understood. Here, we analyzed the phenotype of IRE1α conditional knockout mice and found that IRE1α-deficient mice exhibit mild hypoinsulinemia, hyperglycemia, and a low-weight trend. Moreover, IRE1α disruption causes histological abnormality of the pancreatic acinar and salivary serous tissues and decrease of serum level of immunoglobulin produced in the plasma cells, but not dysfunction of liver. Comparison of this report with previous reports regarding XBP1 conditional knockout mice might provide some clues for the discovery of the novel functions of IRE1α and XBP1.     

La3+ binds to BiP/GRP78 and induces unfolded protein response in HepG2 cells

The effects of La3+ on the unfolded protein response signaling pathways were investigated in human hepatoblastoma HepG2 cells. Our data showed that La3+ could induce unfolded protein response in HepG2 cells, including a significant increase of BiP/GRP78 level, which is an important ER residential chaperone and an ER stress hallmark, in a concentration and time-dependent manner, UPR transducer IRE1 phosphorylation and splicing activation IRE1 downstream substrate XBP1 mRNA. By using La3+-affinity chromatography, the possible cellular target of La3+ leading to UPR events was shown to be the ER residential chaperone BiP/GRP78. BiP/GRP78 was shown to be a La3+ binding protein and the interaction of La3+ with BiP/GRP78 resulted in dissociation of BiP-IRE1 complexes. La3+ induced dissociation of the BiP/GRP78-IRE1 complex was in a time and concentration manner. The apparent dissociation constant was estimated to be 4 nM. In addition, La3+ was observed to slightly stimulate the production of cellular ROS and cause alteration of intracellular Ca2+, indicating the possible involvement of ROS and Ca2+ alteration in La3+ induced UPR. The present work provides a new perspective for understanding the biological and toxicological effects of La3+.     

Role of the unfolded protein response in cell death

Unfolded protein response (UPR) is an important genomic response to endoplasmic reticulum (ER) stress. The ER chaperones, GRP78 and Gadd153, play critical roles in cell survival or cell death as part of the UPR, which is regulated by three signaling pathways: PERK/ATF4, IRE1/XBP1 and ATF6. During the UPR, accumulated unfolded protein is either correctly refolded, or unsuccessfully refolded and degraded by the ubiquitin-proteasome pathway. When the unfolded protein exceeds a threshold, damaged cells are committed to cell death, which is mediated by ATF4 and ATF6, as well as activation of the JNK/AP-1/Gadd153-signaling pathway. Gadd153 suppresses activation of Bcl-2 and NF-κB. UPR-mediated cell survival or cell death is regulated by the balance of GRP78 and Gadd153 expression, which is coregulated by NF-κB in accordance with the magnitude of ER stress. Less susceptibility to cell death upon activation of the UPR may contribute to tumor progression and drug resistance of solid tumors.     

A quantitative method for detection of spliced X-box binding protein-1 (XBP1) mRNA as a measure of endoplasmic reticulum (ER) stress

Endoplasmic reticulum (ER) stress is increasingly recognized as an important mechanism in a wide range of diseases including cystic fibrosis, alpha-1 antitrypsin deficiency, Parkinson's and Alzheimer's disease. Therefore, there is an increased need for reliable and quantitative markers for detection of ER stress in human tissues and cells. Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum can cause ER stress, which leads to the activation of the unfolded protein response (UPR). UPR signaling involves splicing of X-box binding protein-1 (XBP1) mRNA, which is frequently used as a marker for ER stress. In most studies, the splicing of the XBP1 mRNA is visualized by gel electrophoresis which is laborious and difficult to quantify. In the present study, we have developed and validated a quantitative real-time RT-PCR method to detect the spliced form of XBP1 mRNA.     

A modified UPR stress sensing system reveals a novel tissue distribution of IRE1/XBP1 activity during normal Drosophila development

Eukaryotic cells respond to stress caused by the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum by activating the intracellular signaling pathways referred to as the unfolded protein response (UPR). In metazoans, UPR consists of three parallel branches, each characterized by its stress sensor protein, IRE1, ATF6, and PERK, respectively. In Drosophila, IRE1/XBP1 pathway is considered to function as a major branch of UPR; however, its physiological roles during the normal development and homeostasis remain poorly understood. To visualize IRE1/XBP1 activity in fly tissues under normal physiological conditions, we modified previously reported XBP1 stress sensing systems (Souid et al., Dev Genes Evol 217: 159–167, 2007; Ryoo et al., EMBO J 26: 242-252, 2007), based on the recent reports regarding the unconventional splicing of XBP1/HAC1 mRNA (Aragon et al., Nature 457: 736–740, 2009; Yanagitani et al., Mol Cell 34: 191–200, 2009; Science 331: 586–589, 2011). The improved XBP1 stress sensing system allowed us to detect new IRE1/XBP1 activities in the brain, gut, Malpighian tubules, and trachea of third instar larvae and in the adult male reproductive organ. Specifically, in the larval brain, IRE1/XBP1 activity was detected exclusively in glia, although previous reports have largely focused on IRE1/XBP1 activity in neurons. Unexpected glial IRE1/XBP1 activity may provide us with novel insights into the brain homeostasis regulated by the UPR.