Studies on the ubiquitin-like conjugation reaction of Atg8 required for autophagosome formation

　　　Autophagy is a major self-degradative process in eukaryotic cells that playsfundamental roles in cellular and organismal homeostasis, and is involved in manyphysiological and pathological situations. When autophagy is induced, cytoplasmicmaterials and organelles are sequestered into newly emerging double-membranevesicles called autophagosomes, and delivered to the lysosome or the vacuole fordegradation. 　　　In the past decade, many ATG (autophagy-related) genes have been identified bygenetic approaches using the yeast Saccharomyces cerevisiae. Atg8, a ubiquitin-likeprotein (Ubl), is one of the proteins essential for autophagosome formation. The cysteine protease Atg4 first removes the C-terminal arginine of Atg8 to expose the glycine as the new terminus. This glycine forms a thioester bond with Atg7, an activating enzyme (E1), and transfers to and also forms a thioester bond with Atg3, a conjugation enzyme (E2). Atg8 is eventually conjugated to the amino group in the hydrophilic head ofphosphatidylethanolamine (PE). Atg8 is anchored to isolation membrane andautophagosomal membranes probably as this lipid-modified form, and thought to directly participate in the formation of these membranes. Atg4 also catalyzes the deconjugation of Atg8-PE after it has fulfilled its role in autophagosome formation, thus Atg8 is reused. Because the details of this sequential reaction of Atg8 lipidation are unclear, I focus on the mechanism of Atg8 lipidation, in this study. 　　　The Atg8 conjugation system was reconstituted using purified proteins expressed inEscherichia coliand PE-containing liposomes in vitro. First, I successfully captureauthentic thioester intermediates, Atg8-Atg7 and Atg8-Atg3, which can not have beendetected because of their lability. This allows me to analyze the sequential reaction ofAtg8 lipidation. 　　　It was shown that Atg8 could be conjugated with phosphatidylserine (PS) asefficiently as PE in vitro. However, PE was identified as the sole lipid conjugated to theC-terminal glycine of Atg8 in vivo. It suggests that there exists a mechanism that directsAtg8 conjugation preferentially to PE in the cell. In this study, I show that, in contrast toPE conjugation, the PS conjugation of Atg8 is markedly suppressed at physiological(neutral) pH. Then, I show that both of the Atg8-Atg7 and the Atg8-Atg3 intermediatesare formed in the presence of PS liposomes as rapidly as in the presence of PEliposomes, and transfer of Atg8 from Atg3 to PS is specifically retarded at neutral pH. Furthermore, the addition of acidic phospholipids to liposomes is also suggested to result in the preferential formation of the Atg8−PE conjugate. I also show that the acidicphospholipids specifically promote the recruitment of the Atg8-Atg7 and the Atg8-Atg3thioester intermediates to the membrane. Furthermore, .it was reported that theAtg12-Atg5 conjugate, which is formed by ubiquitin-like conjugation reaction, isindispensable for Atg8-PE production in vivo, and that recombinant Atg12-Atg5 indeedstimulates Atg8-PE and Atg8-PS production in vitro. 　　　The preferential formation of Atg8-PE can be achieved by combination of neutralpH, acidic phospholipids, and the Atg12-Atg5 conjugate. Furthermore, I show that PS isnot essential for autophagosome formation even if Atg8 is conjugated to PS in vivo, because the autophagic activity of cells deficient for the PS synthesis enzyme-deficient(pss1△) cells was normal. However, in vitro, the less efficient but significant production of Atg8-PS was still observed, suggesting that the exclusive formation of Atg8-PE requires precise in vivo settings for these factors and/or other facto(s). Alternatively, this result may imply the production of Atg8-PS in vivo, although its amount should be much less than the PE oonjugate. Previously, PE was detected as the sole lipid conjugated to Atg8 and its mammalian homolog LC3 (microtubule-associated protein light chain 3) in vivo. However, lipidated Atg8 and LC3 were forced to accumulate by mutation or treatment with lysosomal inhibitors under nutrient-replete conditions. Therefore, there also remains an alternative possibility that Atg8-PS is formed in starved cells undergoing autophagy, so, I try to purify lipidated Atg8 from the cells under starvation conditions for detailed analysis of lipids conjugated to Atg8 by LC-MS/MS. 　　　Next, I perform gel filtration chromatography to know the interaction of thecomponents in the Atg8 conjugation reaction in vitro. Atg3 interacts with Atg7 as it shown, and Atg3 also interacts with the Atg8-Atg7 thioester intermediate. Furthermore, I find theAtg8-Atg3 thioester intermediate releases from Atg7. It is reasonable because Atg7 isreused rapidly for the next cycle of the reaction. Furthermore, I show that Atg3 and Atg7are interacted via the disulfide bond between active center cysteines in Atg3 and Atg7. Ishow the model of the sequential reaction of Atg8 lipidation

Dietary resveratrol prevents the development of food allergy in mice.

BACKGROUND: Resveratrol is a bioactive polyphenol enriched in red wine that exhibits many beneficial health effects via multiple mechanisms. However, it is unclear whether resveratrol is beneficial for the prevention of food allergy. This study investigated whether resveratrol inhibited the development of food allergy by using a mouse model of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Mice fed standard diet or standard diet plus resveratrol were sensitized by intragastric administration of ovalbumin (OVA) and mucosal adjuvant cholera toxin (CT). Several manifestations of food allergy were then compared between the mice. The effects of resveratrol on T cells or dendritic cells were also examined by using splenocytes from OVA-specific T cell-receptor (TCR) transgenic DO11.10 mice or mouse bone marrow-derived dendritic cells (BMDCs) in vitro. We found that mice fed resveratrol showed reduced OVA-specific serum IgE production, anaphylactic reaction, and OVA-induced IL-13 and IFN-ã production from the mesenteric lymph nodes (MLNs) and spleens in comparison to the control mice, following oral sensitization with OVA plus CT. In addition, resveratrol inhibited OVA plus CT-induced IL-4, IL-13, and IFN-ã production in splenocytes from DO11.10 mice associated with inhibition of GATA-3 and T-bet expression. Furthermore, resveratrol suppressed the OVA plus CT-induced CD25 expression and IL-2 production in DO11.10 mice-splenocytes in association with decreases in CD80 and CD86 expression levels. Finally, resveratrol suppressed CT-induced cAMP elevation in association with decreases in CD80 and CD86 expression levels in BMDCs. CONCLUSIONS/SIGNIFICANCE: Ingestion of resveratrol prevented the development of a food allergy model in mice. Given the in vitro findings, resveratrol might do so by inhibiting DC maturation and subsequent early T cell activation and differentiation via downregulation of CT-induced cAMP activation in mice. These results suggest that resveratrol may have potential for prophylaxis against food allergy

Induction of Colonic Regulatory T Cells by Mesalamine by Activating the Aryl Hydrocarbon ReceptorSummary

Background & Aims: Mesalamine is a first-line drug for treatment of inflammatory bowel diseases (IBD). However, its mechanisms are not fully understood. CD4+ Foxp3+ regulatory T cells (Tregs) play a potential role in suppressing IBD. This study determined whether the anti-inflammatory activity of mesalamine is related to Treg induction in the colon. Methods: We examined the frequencies of Tregs in the colons of wild-type mice, mice deficient for aryl hydrocarbon receptor (AhR-/- mice), and bone marrowâchimeric mice lacking AhR in hematopoietic cells (BM-AhR-/- mice), following oral treatment with mesalamine. We also examined the effects of mesalamine on transforming growth factor (TGF)-β expression in the colon. Results: Treatment of wild-type mice with mesalamine increased the accumulation of Tregs in the colon and up-regulated the AhR target gene Cyp1A1, but this effect was not observed in AhR-/- or BM-AhR-/- mice. In addition, mesalamine promoted in vitro differentiation of naive T cells to Tregs, concomitant with AhR activation. Mice treated with mesalamine exhibited increased levels of the active form of TGF-β in the colon in an AhR-dependent manner and blockade of TGF-β signaling suppressed induction of Tregs by mesalamine in the colon. Furthermore, mice pretreated with mesalamine acquired resistance to dextran sodium sulfateâinduced colitis. Conclusions: We propose a novel anti-inflammatory mechanism of mesalamine for colitis: induction of Tregs in the colon via the AhR pathway, followed by TGF-β activation. Keywords: Mesalamine, Aryl Hydrocarbon Receptor, TGF-β, Regulatory T Cell

Expressions of Tight Junction Proteins Occludin and Claudin-1 Are under the Circadian Control in the Mouse Large Intestine: Implications in Intestinal Permeability and Susceptibility to Colitis

<div><p>Background & Aims</p><p>The circadian clock drives daily rhythms in behavior and physiology. A recent study suggests that intestinal permeability is also under control of the circadian clock. However, the precise mechanisms remain largely unknown. Because intestinal permeability depends on tight junction (TJ) that regulates the epithelial paracellular pathway, this study investigated whether the circadian clock regulates the expression levels of TJ proteins in the intestine.</p><p>Methods</p><p>The expression levels of TJ proteins in the large intestinal epithelium and colonic permeability were analyzed every 4, 6, or 12 hours between wild-type mice and mice with a mutation of a key clock gene Period2 (Per2; mPer2^m/m). In addition, the susceptibility to dextran sodium sulfate (DSS)-induced colitis was compared between wild-type mice and mPer2^m/m mice.</p><p>Results</p><p>The mRNA and protein expression levels of Occludin and Claudin-1 exhibited daily variations in the colonic epithelium in wild-type mice, whereas they were constitutively high in mPer2^m/m mice. Colonic permeability in wild-type mice exhibited daily variations, which was inversely associated with the expression levels of Occludin and Claudin-1 proteins, whereas it was constitutively low in mPer2^m/m mice. mPer2^m/m mice were more resistant to the colonic injury induced by DSS than wild-type mice.</p><p>Conclusions</p><p>Occludin and Claudin-1 expressions in the large intestine are under the circadian control, which is associated with temporal regulation of colonic permeability and also susceptibility to colitis.</p></div

Occludin and Claudin-1 expressions exhibit a time of day-dependent variation in colonic epithelium, relying on normal Per2 activity.

<p>(<b>A</b>) Real-time PCR analysis for Occludin, Zo-1, Claudin-1, -2, -3, -4, -7, and JAM mRNAs expression in the colon tissue samples obtained from the wild-type mice and mPer2^m/m mice at the indicated time points (n = 8 per group). (<b>B</b>) Representative pictures of Western blot for Occludin, Claudin-1 and β-actin expression in colonic epithelial cell samples from wild-type and mPer2^m/m mice obtained at the indicated time points (n = 3 per group). (<b>C</b>) The quantitative analysis of (<b>B</b>) (n = 3 per group). *p<0.05.</p

Clock and Bmal1 bind to the E-box element in the promoter regions of Occludin and Claudin-1 genes and affect their transcriptional responses.

<p>(<b>A</b>) CHiP assays for recruitments of Clock and Bmal1 to E-box elements in Occludin and Claudin-1 promoter region using colonic IECs obtained at the indicated time points from wild-type mice and mPer2^m/m mice (n = 3 per group). Similar results were obtained in two independent experiments. (<b>B</b>) CHiP assays for recruitments of CLOCK and BMAL1 to E-box elements in Occludin and Claudin-1 promoter region using HCT116 cells (n = 3 per group). (<b>C</b>) Relative luciferase activity in HCT116 cells transfected with Occludin and Claudin-1 promoter reporter plasmids together with Clock and Bmal1 expression vectors or a control empty vector (n = 3 per group). *p<0.05.</p

Colonic permeability is under the circadian control.

<p>(<b>A</b>) Permeability to evans blue <i>in vivo</i> at the indicated time points from wild-type mice and mPer2^m/m mice (n = 5 per group). (<b>B</b>) Permeability to FITC-dextran 4000 <i>ex vivo</i> obtained at the indicated time points from wild-type mice and mPer2^m/m mice (n = 6 per group). (<b>C</b>) Permeability to HRP <i>ex vivo</i> obtained at the indicated time points from wild-type mice and mPer2^m/m mice (n = 8 per group). *p<0.05.</p