Boaz Tirosh

Stimulation of Autophagy Improves Endoplasmic Reticulum Stress-Induced Diabetes

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.

The rationale for peptide drug delivery to the colon and the potential of polymeric carriers as effective tools

Abstract The explicit use of colon-specific drug delivery systems is for the local treatment of colon diseases such as ulcerative colitis. Some efficient therapeutic systems, primarily prodrugs and polymeric carriers of salicylate derivatives, have been developed and commercialized during the past 20 years. Speculating that the colon is a superior organ for peptide drug absorption after oral ingestion, many studies indicate that colon-specific drug carriers may potentially be used for the delivery of peptide drugs to that organ. This notion stems from the assumption that the overall proteolytic activity in the colon is lower than and different from the proteolytic activity in the small intestine. For example, it has been found that the degradation rate of albumin, azoalbumin casein, azocasein and collagen in human ileal effluent was faster than the degradation rate in fecal slurries. Other studies, in which the degradation rates of insulin and insulin B-chain in the small and large intestine of the guinea pig were compared, showed higher degradation rates in the small intestine. It is noteworthy, however, that a peptide drug may stay much longer (up to ten times longer) in the large intestine. Thus, even if the enzymic activity is lower, the drug is exposed longer to proteolytic activity. Yet, if the drug is properly protected or formulated with absorption enhancers, the prolonged residence time may increase drug absorption from the large intestine. Thus, prolonged drug blood levels of the ACE inhibitors benazepril and captopril have been demonstrated in a number of studies after colonic administration to rats and dogs. A possible explanation for the `flat pharmacokinetic profiles obtained may be the `closed compartment conditions existing in the colon resulting from the extremely slow propulsive movement of digesta in that organ. These almost stationary conditions may also benefit the performance of functional adjuvants, such as absorption enhancers or peptidase inhibitors, because their dilution rate with the luminal contents of the colon is low. For the purpose of colon-specific drug delivery a variety of polymers has been developed, including acrylic polymers modified with azo cross-linkers and saccharidic polymers. Both kinds have been tested in vitro and in animal studies for their ability to be degraded specifically by typical enzymes of the colon. In addition, swellable polymers were utilized in new pulsatile and delayed-release colonic delivery systems after being protected with enteric coating polymers. To secure peptide drugs in the GI tract, especially in the colon, the use of cross-linked acrylic acid derivatives such as polycarbophil and carbopol® 934 has also been suggested. In conclusion, new biodegradable polymers and polymers with controllable swelling properties can be used for the specific delivery of drugs to the colon. Furthermore, some polymers, by virtue of their intrinsic proteolytic inhibition properties, could be used to improve the absorption of peptide drugs from colonic delivery systems.

Rapid Turnover of Unspliced Xbp-1 as a Factor That Modulates the Unfolded Protein Response

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 ubiquitin-independent 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 non-Xbp-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 Unfolding Is Not a Prerequisite for Endoplasmic Reticulum-to-Cytosol Dislocation

We examined the effects of protein folding on endoplasmic reticulum (ER)-to-cytosol transport (dislocation) by exploiting the well-characterized dihydrofolate reductase (DHFR) domain. DHFR retains the capacity to bind folate analogues in the lumen of microsomes and in the ER of intact cells, upon which it acquires a conformation resistant to proteinase K digestion. Here we show that a Class I major histocompatibility complex heavy chain fused to DHFR is still recognized by the human cytomegalovirus-encoded glycoproteins US2 and US11, resulting in dislocation of the fusion protein from the ERin vitro and in vivo. A folded state of the DHFR domain does not impair dislocation of Class I MHC heavy chainsin vitro or in living cells. In fact, a slight acceleration of the dislocation of DHFR heavy chain fusion was observed in vitro in the presence of a folate analogue. These results suggest that one or more of the channels used for dislocation can accommodate polypeptides that contain a tightly folded domain of considerable size. Our data raise the possibility that the Sec61 channel can be modified to accommodate a folded DHFR domain for dislocation, but not for translocation into the ER, or that a channel altogether distinct from Sec61 is used for dislocation.

HCV Causes Chronic Endoplasmic Reticulum Stress Leading to Adaptation and Interference with the Unfolded Protein Response

Background The 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 Findings The 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. Conclusions Our 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.

Mucus Gel Thickness and Turnover in the Gastrointestinal Tract of the Rat: Response to Cholinergic Stimulus and Implication for Mucoadhesion

The thickness of the mucus gel and its turnover rate were measured in the stomach, proximal jejunum, cecum and proximal colon of the rat, using microscopy and staining techniques. The specific mucus-secretory responses to carbachol-induced cholinergic stimulus in these locations were also studied. The mucus gel was found to be the thinnest (18 ±1 microns) in the cecum, and the thickest in the stomach (39 ±14 microns). The effect of carbachol on mucus secretion was profound and dose dependent in the stomach, and less profound, although still dose dependent, in the proximal jejunum. The least responsive organs were the cecum and the proximal colon, where no effect was observed after three doses of carbachol. Mucus secretion rate was significantly higher in the jejunum (1.1 ± 0.5 µg glucose equivalent min−1 cm−2) than in the colon (0.5 ± 0.2 µg glucose equivalent min−l cm−2). Also, the proximal jejunum was more responsive to the carbachol stimulus (mucus secretion rate of 5.4 ±2.2 µg glucose equivalent min−1 cm−2 after carbachol treatment) than the colon (mucus secretion rate of 1.0 ±0.4 µg glucose equivalent min−l cm−2 after carbachol treatment). In vitro mucoadhesion studies with Polycarbophil disks were performed in the mucosal tissues of the stomach, jejunum, cecum and proximal colon of the rat with and without cholinergic (carbachol) stimulus. The adhesion force in the cecum and the colon was significantly stronger than in the stomach and proximal jejunum when the studies were performed at pH 2. Carbachol treatment did not significantly change the mucoadhesion of Polycarbophil disks. It is concluded that in the gastrointestinal tract of the rat the colon and the cecum are more suitable locations for the mucoadhesion than the stomach and the jejunum because: (1) their mucus turnover is lower, (2) their sensitivity to mucus secretory stimulus is lower, and (3) their Polycarbophil adherence properties are stronger.

CHOP is a critical regulator of acetaminophen-induced hepatotoxicity.

BACKGROUND & AIMSnThe liver is a major site of drug metabolism and elimination and as such is susceptible to drug toxicity. Drug induced liver injury is a leading cause of acute liver injury, of which acetaminophen (APAP) is the most frequent causative agent. APAP toxicity is initiated by its toxic metabolite NAPQI. However, downstream mechanisms underlying APAP induced cell death are still unclear. Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have recently emerged as major regulators of metabolic homeostasis. UPR regulation of the transcription repressor CHOP promotes cell death. We analyzed the role of UPR and CHOP in mediating APAP hepatotoxicity.nnnMETHODSnA toxic dose of APAP was orally administered to wild type (wt) and CHOP knockout (KO) mice and damage mechanisms were assessed.nnnRESULTSnCHOP KO mice were protected from APAP induced damage and exhibited decreased liver necrosis and increased survival. APAP metabolism in CHOP KO mice was undisturbed and glutathione was depleted at similar kinetics to wt. ER stress and UPR activation were overtly seen 12h following APAP administration, a time that coincided with strong upregulation of CHOP. Remarkably, CHOP KO but not wt mice exhibited hepatocyte proliferation at sites of necrosis. In vitro, large T immortalized CHOP KO hepatocytes were protected from APAP toxicity in comparison to wt control cells.nnnCONCLUSIONSnCHOP upregulation during APAP induced liver injury compromises hepatocyte survival in various mechanisms, in part by curtailing the regeneration phase following liver damage. Thus, CHOP plays a pro-damage role in response to APAP intoxication.

XBP-1 specifically promotes IgM synthesis and secretion, but is dispensable for degradation of glycoproteins in primary B cells

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.

Human Cytomegalovirus Protein US11 Provokes an Unfolded Protein Response That May Facilitate the Degradation of Class I Major Histocompatibility Complex Products

ABSTRACT The human cytomegalovirus (HCMV) glycoprotein US11 diverts class I major histocompatibility complex (MHC) heavy chains (HC) from the endoplasmic reticulum (ER) to the cytosol, where HC are subjected to proteasome-mediated degradation. In mouse embryonic fibroblasts that are deficient for X-box binding protein 1 (XBP-1), a key transcription factor in the unfolded protein response (UPR) pathway, we show that degradation of endogenous mouse HC is impaired. Moreover, the rate of US11-mediated degradation of ectopically expressed HLA-A2 is reduced when XBP-1 is absent. In the human astrocytoma cell line U373, turning on expression of US11, but not US2, is sufficient to induce a UPR, as manifested by upregulation of the ER chaperone Bip and by splicing of XBP-1 mRNA. In the presence of dominant-negative versions of XBP-1 and activating transcription factor 6, the kinetics of class I MHC HC degradation were delayed when expression of US11 was turned on. The magnitude of these effects, while reproducible, was modest. Conversely, in cells that stably express high levels of US11, the degradation of HC is not affected by the presence of the dominant negative effectors of the UPR. An infection of human foreskin fibroblasts with human cytomegalovirus induced XBP-1 splicing in a manner that coincides with US11 expression. We conclude that the contribution of the UPR is more pronounced on HC degradation shortly after induction of US11 expression and that US11 is sufficient to induce such a response.

ER stress and its regulator X-box binding protein-1 enhance polyIC induced innate immune response in dendritic cells

Multiple physiological and pathological conditions interfere with the function of the endoplasmic reticulum (ER). However, much remains unknown regarding the impact of ER stress on inflammatory responses in dentritic cells (DCs) upon the recognition of pathogen molecules. We show that ER stress greatly potentiates the expression of inflammatory cytokines and IFN‐β in murine DCs stimulated by polyIC, a synthetic mimic of virus dsRNA. Both toll‐like receptor 3 and melanoma differentiation‐associated gene‐5 are involved in the enhanced IFN‐β production, which is associated with increased activation of NF‐κB and IRF3 signaling as well as the splicing of X‐box‐binding protein‐1 (XBP‐1), an important regulator involved in ER stress response. Surprisingly, silencing of XBP‐1 reduces polyIC‐stimulated IFN‐β expression in the presence or absence of ER stress, indicating that XBP‐1 may be essential for polyIC signaling and ER stress‐amplified IFN‐β production. Overexpression of a spliced form of XBP‐1 (XBP‐1s) synergistically augments polyIC‐induced inflammatory response. For the first time, we show that XBP‐1s overexpression‐enhanced IFN‐β production in DCs markedly suppresses vesicular stomatitis virus infection, revealing a previously unrecognized role for XBP‐1 in an antiviral response. Our findings suggest that evolutionarily conserved ER stress response and XBP‐1 may function collaboratively with innate immunity to maintain cellular homeostasis.