Function of yeast and amphioxus tRNA ligase in IRE1alpha-dependent XBP1 mRNA splicing

During their maturation step, transfer RNAs (tRNAs) undergo excision of their introns by specific splicing. Although tRNA splicing is a molecular event observed in all domains of life, the machinery of the ligation reaction has diverged during evolution. Yeast tRNA ligase 1 (TRL1) is a multifunctional protein that alone catalyzes RNA ligation in tRNA splicing, whereas three molecules [RNA ligase (RNL), Clp1, and PNK/CPDase] are necessary for RNA ligation in tRNA splicing in amphioxi. RNA ligation not only occurs in tRNA splicing, but also in yeast HAC1 mRNA splicing and in animal X-box binding protein 1 (XBP1) mRNA splicing under conditions of endoplasmic reticulum (ER) stress. Yeast TRL1 is known to function as an RNA ligase for HAC1 mRNA splicing, whereas the RNA ligase for XBP1 mRNA splicing is unknown in animals. We examined whether yeast and amphioxus RNA ligases for tRNA splicing function in RNA ligation in mammalian XBP1 splicing. Both RNA ligases functioned in RNA ligation in mammalian XBP1 splicing in vitro. Interestingly, Clp1, and PNK/CPDase were not necessary for exon–exon ligation in XBP1 mRNA by amphioxus RNL. These results suggest that RNA ligase for tRNA splicing might therefore commonly function as an RNA ligase for XBP1 mRNA splicing.     

RNF13通过IRE1/XBP-1 通路调控内质网应激介导的细胞凋亡

目的:在研究内质网应激介导的细胞凋亡过程中,我们发现Ring finger protein13(RNF13)具有促进细胞凋亡的功能。我们拟研究沉默RNF13后细胞对Tunicamycin等引起的细胞凋亡的影响,以及RNF13对活性形式的caspase3,XBP1(X-box binding protein 1)的剪切以及IRE1(Endoplasmic reticulum to nucleus signaling 1)磷酸化的影响以有助于了解RNF13促进细胞凋亡的信号通路的研究。方法:基因沉默RNF13,利用MTT方法研究RNF13沉默后对细胞增殖的影响,RNF13基因沉默后对XBP1剪切的影响,免疫印迹观察RNF13对IRE1磷酸化的影响。结果:RNF13基因沉默效率在80%以上。RNF13基因沉默后明显抑制细胞凋亡;敲低RNF13的细胞可抵抗衣霉素以及毒胡萝卜素的诱导的细胞凋亡。Caspase-3是细胞凋亡的关键蛋白。敲低RNF13后caspase-3的活性形式明显降低(降低70%,P0.001)。在加入衣霉素引起内质网应激的情况下,敲除RNF13的细胞XBP1的切割活性明显降低。敲除RNF13的细胞中IREl的磷酸化明显降低(降低90%,P0.001)。结论:RNF13通过IRE1-XBP1信号通路调节细胞凋亡。     

Reactive oxygen species‐mediated endoplasmic reticulum stress response induces apoptosis of Mycobacterium avium‐infected macrophages by activating regulated IRE1‐dependent decay pathway

Mycobacterium avium, a slow‐growing nontuberculous mycobacterium, causes fever, diarrhoea, loss of appetite, and weight loss in immunocompromised people. We have proposed that endoplasmic reticulum (ER) stress‐mediated apoptosis plays a critical role in removing intracellular mycobacteria. In the present study, we investigated the role of the regulated IRE1‐dependent decay (RIDD) pathway in macrophages during M. avium infection based on its role in the regulation of gene expression. The inositol‐requiring enzyme 1 (IRE1)/apoptosis signal‐regulating kinase 1 (ASK1)/c‐Jun N‐terminal kinase (JNK) signalling pathway was activated in macrophages after infection with M. avium. The expression of RIDD‐associated genes, such as Bloc1s1 and St3gal5, was decreased in M. avium‐infected macrophages. Interestingly, M. avium‐induced apoptosis was significantly suppressed by pretreatment with irestatin (inhibitor of IRE1α) and 4μ8c (RIDD blocker). Macrophages pretreated with N‐acetyl cysteine (NAC) showed decreased levels of reactive oxygen species (ROS), IRE1α, and apoptosis after M. avium infection. The expression of Bloc1s1 and St3gal5 was increased in NAC‐pretreated macrophages following infection with M. avium. Growth of M. avium was significantly increased in irestatin‐, 4μ8c‐, and NAC‐treated macrophages compared with the control. The data indicate that the ROS‐mediated ER stress response induces apoptosis of M. avium‐infected macrophages by activating IRE1α‐RIDD. Thus, activation of IRE1α suppresses the intracellular survival of M. avium in macrophages.     

Degradation of the endoplasmic reticulum by autophagy in plants

Eukaryotic cells have developed sophisticated strategies to contend with environmental stresses faced in their lifetime. Endoplasmic reticulum (ER) stress occurs when the accumulation of unfolded proteins within the ER exceeds the folding capacity of ER chaperones. ER stress responses have been well characterized in animals and yeast, and autophagy has been suggested to play an important role in recovery from ER stress. In plants, the unfolded protein response signaling pathways have been studied, but changes in ER morphology and ER homeostasis during ER stress have not been analyzed previously. Autophagy has been reported to function in tolerance of several stress conditions in plants, including nutrient deprivation, salt and drought stresses, oxidative stress, and pathogen infection. However, whether autophagy also functions during ER stress has not been investigated. The goal of our study was to elucidate the role and regulation of autophagy during ER stress in Arabidopsis thaliana.     

Polystyrene nanoplastics aggravates lipopolysaccharide-induced apoptosis in mouse kidney cells by regulating IRE1/XBP1 endoplasmic reticulum stress pathway via oxidative stress

Nanoplastics (NPs) pollution poses a huge threat to the ecosystem and has become one of the environmental pollutants that have attracted much attention. There is increasing evidence that both oxidative stress and endoplasmic reticulum stress (ERS) are associated with polystyrene nanoplastics (PS-NPs) exposure. Lipopolysaccharide (LPS) has been shown to induce apoptotic damage in various tissues, but whether PS-NPs can aggravate LPS-induced apoptosis in mouse kidneys through oxidative stress-regulated inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) ERS pathway remains unclear. In this study, based on the establishment of in vitro and in vivo PS-NPs and LPS exposure models alone and in combination in mice and HEK293 cells, the effects and mechanisms of PS-NPs on LPS-induced renal cell apoptosis were investigated. The results showed that PS-NPs could aggravate LPS-induced apoptosis. PS-NPs/LPS can induce ERS through oxidative stress, activate the IRE1/XBP1 pathway, and promote the expression of apoptosis markers (Caspase-3 and Caspase-12). Kidney oxidative stress, ERS, and apoptosis in PS-NPs + LPS combined exposure group were more severe than those in the single exposure group. Interestingly, 4-phenylbutyric acid-treated HEK293 cells inhibited the expression of the IRE1/XBP1 ERS pathway and apoptotic factors in the PS-NPs + LPS combined exposure group. N-acetyl-L-cysteine effectively blocked the activation of the IRE1/XBP1 ERS pathway, suggesting that PS-NPs-induced oxidative stress is an early event that triggers ERS. Collectively, these results confirmed that PS-NPs aggravated LPS-induced apoptosis through the oxidative stress-induced IRE1/XBP1 ERS pathway. Our study provides new insights into the health threats of PS-NPs exposed to mammals and humans.     

Beta Cell Dedifferentiation Induced by IRE1α Deletion Prevents Type 1 Diabetes

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CD437 induces apoptosis in ovarian adenocarcinoma cells via ER stress signaling

A synthetic retinoid, CD437, has been shown to exert potent anti-tumor activity against various types of cancer cell lines, regardless of their sensitivities to natural retinoids. We herein demonstrate that CD437 induces endoplasmic reticulum (ER) stress, including the up-regulation of CHOP, BIP and GADD34 mRNA through ER stress transducer (PERK and IRE1α) activation in an ovarian adenocarcinoma cell line, SKOV3. It was also shown that CD437 induced the CHOP and GADD34 expressions in another four ovarian adenocarcinoma cell lines, indicating that CD437 functions as an ER stress inducer in these cell lines. Moreover, the siRNA-mediated knockdown of inducible CHOP expression prevented the cytotoxic effect of CD437. These results suggest that ER stress plays an important role in the mechanism by which CD437 induces apoptosis in ovarian adenocarcinoma cells.     

Pre-sorting endosomal transport of the GPI-anchored protein, CD59, is regulated by EHD1

EHD1 regulates the trafficking of multiple receptors from the endocytic recycling compartment (ERC) to the plasma membrane. However, the potential role of EHD1 in regulating the family of glycosylphosphatidylinositol-anchored proteins (GPI-APs) has not been determined. Here we demonstrate a novel role for EHD1 in regulating the trafficking of CD59, an endogenous GPI-AP, at early stages of trafficking through the endocytic pathway. EHD1 displays significant colocalization with newly internalized CD59. Upon EHD1 depletion, there is a rapid Rab5-independent coalescence of CD59 in the ERC region. However, expression of an active Arf6 mutant (Q67L), which traps internalized pre-sorting endosomal cargo in phosphatidylinositol(4,5)-bisphosphate enriched vacuoles, prevents this coalescence. It is of interest that sustained PKC activation leads to a similar coalescence of CD59 at the ERC, and treatment of EHD1-depleted cells with a PKC inhibitor (Go6976) blocked this rapid relocation of CD59. However, unlike sustained PKC activation, EHD1 depletion does not induce the translocation of PKCα to ERC. The results presented herein provide evidence that EHD1 is involved in the control of CD59 transport from pre-sorting endosomes to the ERC in a PKC-dependent manner. However, the mechanisms of EHD1-induced coalescence of CD59 at the ERC differ from those induced by sustained PKC activation.     

Ubiquitination of Inositol-requiring Enzyme 1 (IRE1) by the E3 Ligase CHIP Mediates the IRE1/TRAF2/JNK Pathway

Deciphering the inositol-requiring enzyme 1 (IRE1) signaling pathway is fundamentally important for understanding the unfolded protein response (UPR). The ubiquitination of proteins residing on the endoplasmic reticulum (ER) membrane has been reported to be involved in the UPR, although the mechanism has yet to be fully elucidated. Using immunoprecipitation and mass spectrometry, IRE1 was identified as a substrate of the E3 ligase CHIP (carboxyl terminus of HSC70-interacting protein) in HEK293 cells under geldanamycin-induced ER stress. Two residues of IRE1, Lys⁵⁴⁵ and Lys⁸²⁸, were targeted for Lys⁶³-linked ubiquitination. Moreover, in CHIP knockdown cells, IRE1 phosphorylation and the IRE1-TRAF2 interaction were nearly abolished under ER stress, which may be due to lacking ubiquitination of IRE1 on Lys⁵⁴⁵ and Lys⁸²⁸, respectively. The cellular responses were evaluated, and the data indicated that CHIP-regulated IRE1/TRAF2/JNK signaling antagonized the senescence process. Therefore, our findings suggest that CHIP-mediated ubiquitination of IRE1 contributes to the dynamic regulation of the UPR.     

CD59、CD55在T细胞活化信号转导中的协同作用

目的:研究糖基磷脂酰肌醇(GPI)锚固蛋白CD59、CD55在脂筏介导T细胞信号转导通路中的协同效应。方法:应用siRNA技术,构建特异性针对CD55与CD59基因的重组载体pSUPER-siCD55,pSUPER-siCD59。实验分为未转染的Jurkat细胞组(Ⅰ组)、转染pSUPER空质粒的Jurkat细胞组(Ⅱ组)、转染pSUPER-siCD59重组质粒的Jurkat细胞组(Ⅲ组)及转染pSUPER-siCD55重组质粒的Jurkat细胞组(Ⅳ组)。RT-PCR检测转染细胞中CD55和CD59基因的表达噻唑蓝(MTT)比色法和激光共聚焦扫描显微镜分别检测CD55与CD59联合作用对4组Jurkat细胞的增殖效应以及细胞内钙离子的变化、结果:稳定转染后,Ⅲ组细胞CD59分子的表达和Ⅳ组细胞CD55分子的表达被成功抑制。Ⅰ组和Ⅱ组细胞CD55与CD59联合作用后增殖能力和钙离子浓度均明显高于Ⅲ组、Ⅳ组(P<0.05),Ⅰ组和Ⅱ组之间无差异结论:CD59和CD55在T细胞活化信号转导通路中存在协同效应。     

CD59、CD55在T细胞活化信号转导中的协同作用

目的：研究糖基磷脂酰肌醇（GeD）锚固蛋白CD59、CD55在脂筏介导T细胞信号转导通路中的协同效应。方法：应用siRNA技术,构建特异性针对CD55与CD59基因的重组载体pSUPER—siCD55,pSUPER—siCD59。实验分为未转染的Jurkat细胞组（I组）、转染pSUPER空质粒的Jurkat细胞组（Ⅱ组）、转染pSUPER—siCD59重组质粒的Jurkat细胞组（Ⅲ组）及转染pSUPER—siCD55重组质粒的Jurkat细胞组（Ⅳ组）。RT—PCR检测转染细胞中CD55和CD59基因的表达。噻唑蓝（MTT）比色法和激光共聚焦扫描显微镜分别检测CD55与CD59联合作用对4组Jurkat细胞的增殖效应以及细胞内钙离子的变化。结果：稳定转染后,Ⅲ组细胞CD59分子的表达和Ⅳ组细胞CD55分子的表达被成功抑制。Ⅰ组和Ⅱ组细胞CD55与CD59联合作用后增殖能力和钙离子浓度均明显高于Ⅲ组、Ⅳ组（P〈0．05）,Ⅰ组和Ⅱ组之间无差异。结论：CD59和CD55在T细胞活化信号转导通路中存在协同效应。