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Accumulation of misfolded proinsulin in the β-cell leads to dysfunction induced by endoplasmic reticulum (ER) stress, with diabetes as a consequence. Autophagy helps cellular adaptation to stress via clearance of misfolded proteins and damaged organelles. We studied the effects of proinsulin misfolding on autophagy and the impact of stimulating autophagy on diabetes progression in Akita mice, which carry a mutation in proinsulin, leading to its severe misfolding. Treatment of female diabetic Akita mice with rapamycin improved diabetes, increased pancreatic insulin content, and prevented β-cell apoptosis. In vitro, autophagic flux was increased in Akita β-cells. Treatment with rapamycin further stimulated autophagy, evidenced by increased autophagosome formation and enhancement of autophagosome–lysosome fusion. This was associated with attenuation of cellular stress and apoptosis. The mammalian target of rapamycin (mTOR) kinase inhibitor Torin1 mimicked the rapamycin effects on autophagy and stress, indicating that the beneficial effects of rapamycin are indeed mediated via inhibition of mTOR. Finally, inhibition of autophagy exacerbated stress and abolished the anti-ER stress effects of rapamycin. In conclusion, rapamycin reduces ER stress induced by accumulation of misfolded proinsulin, thereby improving diabetes and preventing β-cell apoptosis. The beneficial effects of rapamycin in this context strictly depend on autophagy; therefore, stimulating autophagy may become a therapeutic approach for diabetes.

Natural killer (NK) cells mediate innate immune responses against hazardous cells and are particularly important for the control of human cytomegalovirus (HCMV). NKG2D is a key NK activating receptor that recognizes a family of stress-induced ligands, including MICA, MICB, and ULBP1-6. Notably, most of these ligands are targeted by HCMV proteins and a miRNA to prevent the killing of infected cells by NK cells. A particular highly prevalent MICA allele, MICA008, is considered to be an HCMV-resistant "escape variant" that confers advantage to human NK cells in recognizing infected cells. However, here we show that HCMV uses its viral glycoprotein US9 to specifically target MICA008 and thus escapes NKG2D attack. The finding that HCMV evolved a protein dedicated to countering a single host allele illustrates the dynamic co-evolution of host and pathogen.

The mammalian and yeast unfolded protein responses (UPR) share the characteristic of rapid elimination of unspliced Xbp-1 (Xbp-1u) and unspliced Hac1p, respectively. These polypeptides derive from mRNAs, whose splicing is induced upon onset of the UPR, so as to allow synthesis of transcription factors essential for execution of the UPR itself. Whereas in yeast translation of unspliced Hac1p is blocked, mammalian Xbp-1u is synthesized constitutively and eliminated by rapid proteasomal degradation. Here we show that the rate of Xbp-1u degradation approaches its rate of synthesis. The C terminus of XBP-1u ensures its trafficking to the cytoplasm, and is sufficient to impose rapid degradation. Degradation of XBP-1u involves both ubiquitin-dependent and ubiquitinindependent mechanisms, which might explain its unusually rapid turnover. Xbp-1 ؊/؊ mouse embryonic fibroblasts reconstituted with mutants of XBP-1u that show improved stability differentially activate UPR target genes. Unexpectedly, we found that one of the mutants activates transcription of both Xbp-1-specific and nonXbp-1-dependent UPR targets in response to tunicamycin treatment, even more potently than does wild type Xbp-1. We suggest that the degradation of Xbp-1u is required to prevent uncontrolled activation of the UPR while allowing short dwell times for initiation of this response.Protein folding in the endoplasmic reticulum (ER) 4 is carried out under the constant scrutiny of the ER quality control machinery (1). The overall capacity of the ER to fold newly synthesized proteins must match the load of client proteins that emerge into the ER. When this amount exceeds the folding capacity of the ER, a signaling pathway emanates from the ER that controls gene transcription, as well as protein translation. This ER to nucleus signaling cascade is referred to as the unfolded protein response (UPR). The overall goal of the UPR is to enhance the clearance of misfolded proteins from the ER, and consequently the UPR alleviates ER stress (2).In yeast, Ire1p is the only known transducer of the UPR. In response to ER stress conditions, Ire1p dimerizes and undergoes autophosphorylation. This event induces a conformational change that activates a nuclease domain located in its cytosolic tail (3, 4). By means of this nuclease activity, activated Ire1p splices the mRNA of Hac1, which in its spliced form encodes Hac1p, a potent transcription factor that induces transcription of many genes that encode ER chaperones, proteins that participate in ER to Golgi trafficking and components of the ER degradation machinery (2).The mammalian UPR is minimally composed of three transducers: Perk, Atf6, and Ire1 (5). Ire1 is highly conserved from yeast to mammals, but the homolog of Hac1 eluded scientists for many years. The mammalian counterpart of Hac1 was identified as Xbp-1. Xbp-1, a member of the CREB/ATF family of transcription factors, does not share any significant sequence homology with Hac1. It is composed of a basic leucine zipper-containing DNA binding domain located a...

Inositol-requiring enzyme 1 (IRE1) is a kinase and ribonuclease that executes the splicing of X box binding protein 1 (XBP-1) mRNA in response to the accumulation of unfolded protein in the ER, a signal cascade termed the unfolded protein response. Recently, IRE1 has been implicated in mRNA and miRNA cleavage and degradation, a pathway termed regulated IRE1-dependent decay (RIDD). Deletion of XBP-1 in the liver and pancreas strongly enhances RIDD by upregulating IRE1 protein levels and enhancing its ribonuclease activity. Because XBP-1 is essential for generating plasma cells with developed secretory capacity, we sought to evaluate the contribution of RIDD to this regulation. Mice were conditionally deleted for XBP-1 and/or IRE1 in their B-cell lineage. Similarly to the liver, deletion of XBP-1 induces IRE1 expression in LPS-treated B cells. In vitro, IRE1 cleaves the mRNA of secretory μ chains, which explains the reduction in secretory μ mRNA and its synthesis in XBP-1 KO plasma cells. In accordance, the IgM response is partially restored in XBP-1/IRE1 double KO mice relative to XBP-1 KO mice. Interestingly, the IgG1 response is reduced to a similar level in XBP-1 KO, IRE1 KO, and their double knockout animals. Our data demonstrate a specific contribution by RIDD in curtailing immunoglobulin synthesis and secretion.Keywords: ER stress r IRE1 r plasma cells r RIDD r UPR r XBP-1 See accompanying Commentary by van Anken et al.Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe unfolded protein response (UPR) is an adaptive signaling pathway elicited in response to perturbations in ER homeostasis, conditions referred to as ER stress. In the canonical mammalian UPR, three ER stress sensors operate in parallel: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase, and activating transcripCorrespondence: Dr. Boaz Tirosh e-mail: boazt@ekmd.huji.ac.il tion factor 6 (reviewed in [1]). Upon activation, each sensor arm executes a transcriptional program that is interconnected with the other UPR pathways for amplification and negative feedbacks. Thus, the outcome of the UPR is dependent on the relative contribution of the three arms and their threshold for activation. Much of the molecular details of UPR signaling were brought about using artificial induction with chemicals that robustly perturb protein folding in the ER, such as tunicamycin, thapsigargin, or DTT. Understanding of the UPR in a physiological setting, however, has been more challenging to elucidate.C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 868Sandrine Benhamron et al. Eur. J. Immunol. 2014. 44: 867-876 The first example for a physiological role of the UPR was described in the terminal differentiation of mature B cells into plasma cells (PCs), the latter are responsible for secretion of antibodies [2]. Mature B cells contain very little cytoplasm and intracellular membranes. When triggered by antigen in the context of accessory signals such as toll-lik...

The need for improved specificity in the local treatment of inflammatory bowel diseases (IBD) led us to use negatively charged liposomes to target the inflamed colonic epithelium. The purpose of the present study was to elucidate the cause for our previous observations that such liposomes accumulate, preferentially, in the inflamed mucosa of rats that were induced with experimental colitis, following luminal administration. Protein analysis (tandem mass spectrometry, verified by Western blot) of inflamed mucosal specimens, extracted at pH 3, 5 and 7, revealed an increased expression of transferrin (TF) at pH 3. Histological examination indicated that the TF was located at the luminal side of the inflamed epithelium. Negatively charged (but not neutral) liposomes adhered to both commercial and mucosal TF at low pH, but not at neutral pH. Moreover, preincubation of negatively charged liposomes with TF profoundly attenuated their adherence to the inflamed mucosa of the rat colon. It is concluded that, at a low pH, typical of the colon lumen in ulcerative colitis, TF mediates specific mucoadhesion of negatively charged liposomes to the inflamed mucosa. This observation could be useful in the rational design of specific drug vehicles aimed at IBD therapy after luminal administration.

BackgroundThe endoplasmic reticulum (ER) is the cellular site for protein folding. ER stress occurs when protein folding capacity is exceeded. This stress induces a cyto-protective signaling cascades termed the unfolded protein response (UPR) aimed at restoring homeostasis. While acute ER stress is lethal, chronic sub-lethal ER stress causes cells to adapt by attenuation of UPR activation. Hepatitis C virus (HCV), a major human pathogen, was shown to cause ER stress, however it is unclear whether HCV induces chronic ER stress, and if so whether adaptation mechanisms are initiated. We wanted to characterize the kinetics of HCV-induced ER stress during infection and assess adaptation mechanisms and their significance.Methods and FindingsThe HuH7.5.1 cellular system and HCV-transgenic (HCV-Tg) mice were used to characterize HCV-induced ER stress/UPR pathway activation and adaptation. HCV induced a wave of acute ER stress peaking 2–5 days post-infection, which rapidly subsided thereafter. UPR pathways were activated including IRE1 and EIF2α phosphorylation, ATF6 cleavage and XBP-1 splicing. Downstream target genes including GADD34, ERdj4, p58ipk, ATF3 and ATF4 were upregulated. CHOP, a UPR regulated protein was activated and translocated to the nucleus. Remarkably, UPR activity did not return to baseline but remained elevated for up to 14 days post infection suggesting that chronic ER stress is induced. At this time, cells adapted to ER stress and were less responsive to further drug-induced ER stress. Similar results were obtained in HCV-Tg mice. Suppression of HCV by Interferon-α 2a treatment, restored UPR responsiveness to ER stress tolerant cells.ConclusionsOur study shows, for the first time, that HCV induces adaptation to chronic ER stress which was reversed upon viral suppression. These finding represent a novel viral mechanism to manipulate cellular response pathways.

Differentiation of B cells into plasma cells requires X-box binding protein–1 (XBP-1). In the absence of XBP-1, B cells develop normally, but very little immunoglobulin is secreted. XBP-1 controls the expression of a large set of genes whose products participate in expansion of the endoplasmic reticulum (ER) and in protein trafficking. We define a new role for XBP-1 in exerting selective translational control over high and sustained levels of immunoglobulin M (IgM) synthesis. XBP-1−/− and XBP-1+/+ primary B cells synthesize IgM at comparable levels at the onset of stimulation with lipopolysaccharide or CpG. However, later there is a profound depression in synthesis of IgM in XBP-1−/− B cells, notwithstanding similar levels of μmRNA. In marked contrast, lack of XBP-1 does not affect synthesis and trafficking of other glycoproteins, or of immunoglobulin light chains. Contrary to expectation, degradation of proteins from the ER, using TCRα or US11-mediated degradation of class I major histocompatibility complex molecules as substrates, is normal in XBP-1−/− B cells. Furthermore, degradation of membrane μ was unaffected by enforced expression of XBP-1. We conclude that in primary B cells, the XBP-1 pathway promotes synthesis and secretion of IgM, but does not seem to be involved in the degradation of ER proteins, including that of μ chains themselves.