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RNF2Interacts with MANF and Protects Neurons from ER Stress-induced Injury

Author: ChenZuo
Tutor: ShenYuXian
School: Anhui Medical University,
Course: Pharmacology
Keywords: Yeast two-hybrid MANF RNF2 neural protection
CLC: R969.2
Type: Master's thesis
Year: 2013
Downloads: 14
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Abstract


Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an evolutionarilyconserved neurotrophic factor. It is an ER stress-inducible protein. MANF has beenproved to protect neurons from different pathological insults. However, the underlyingmechanisms are not fully understood. To provide new clues for exploring the functionof MANF in neuron protection, we performed yeast two-hybrid screening to identifyproteins interacting with MANF using human fetal brain library. Seven novel MANFinteracting proteins including Homo sapiens RNF2were obtained. The interaction ofMANF and RNF2was confirmed by in vitro glutathione S-transferase pull-down andin vivo co-immunoprecipitation.RNF2, a component of the polycomb group (PcG) proteins, is highly expressed in many tumors. It has been previously reported that the PcG proteins SCMH1or BMI1could induce tolerance to ischemia and RNF2increased in ischemic brain and retina.However, whether RNF2also has neuron protection is still unknown. In this study, weobserved the expression of RNF2and MANF in cerebral ischemia. We also detectedthe effect of RNF2overexpression or knockdown on N2A cells treated with TM.Objective:To screen proteins interacting with MANF using yeast two-hybrid system, and confirmthe interaction between MANF and RNF2. We also aim to investigate whether RNF2has the protective effects on neural cells.Methods:The interactions between MANF and the host cell proteins were analyzed using a yeasttwo-hybrid technique. The cMyc-MANF fusion protein expressed by pGBKT7plasmid in Gold yeast was bound to host cell proteins from a human fetal brain cDNAlibrary transformed to AH109yeast using a mating method. The interaction betweenMANF and RNF2was confirmed by glutathione S-transferase pull-down in vitro andco-immunoprecipitation in vivo. The yeast co-transformation was used to identify theprotein interaction in the yeast. Immunofluorescent Staining was performed to assessthe localization of MANF and RNF2with or without the treatment of tunicamycin (TM).To figure the location where MANF interacts with RNF2, the fractions of nucleus andcytosol of N2A cells were isolated. We established N2A-MANF cell line stablyexpressing MANF. The pEGFP-C2-MANF was transfected into N2A cells bylipofectamine and the positive clones were screened by G418. The expression ofMANF in positive clones was detected by fluorescence microscope, RT-PCR, and western blotting. Focal cerebral ischemic model induced by middle cerebral arteryocclusion (MCAO) was established by inserting a nylon monofilament through theright common carotid artery into the anterior cerebral artery. The filament waswithdrawn at2hrs after occlusion to allow reperfusion for24hrs or48hrs. Doublelabeled immunofluorescent staining was used to investigate the expression anddistribution of MANF and RNF2in the cerebral cortex after MCAO. Afterover-expression or knockdown RNF2gene in N2A cells, PI staining was used toidentify the dead neural cells; western blotting was used to determine the expression ofcleaved caspase-3and CHOP with or without TM treatment.Results:1. Screening proteins interacting with MANF by using yeast two-hybrid system1.1Determination of autonomous activation of the bait protein MANFWe constructed pGBKT7-MANF as a bait plasmid. Using LiAc-mediated yeasttransformation, the bait plasmid pGBKT7-MANF was transformed into the yeast strainGold to produce the Gold/pGBKT7-MANF cell line. It showed that bait culturesgrown in the liquid YPDA didn’t grow more slowly than the control.Gold/pGBKT7-MANF grew well on the SD/-Trp plate, but did not grow on the DDOplate neither turned blue on the SD/-Trp/X-α-gal plate. This suggests that MANF as abait protein may not have the toxicity and autonomous activation.1.2Expression of MANF fusion protein in Gold yeastWestern blotting showed that the Gold yeast strain transformed with pGBKT7-MANFexpressed the fusion protein at high level with about45kD relative molecular weight.The Gold/pGBKT7yeast cells used as the positive control expressed a25kD protein,but the untransformed yeast Gold cells used as the negative control did not express the fusion protein. Therefore, the transformed yeast was specific and able to be used foryeast two-hybrid analysis.1.3Screening the human fetal brain cDNA library for MANF interacting proteinsThe yeast strain Gold/pGBKT7-MANF was mated with the Mate&PlateTMHumanFetal Brain cDNA library.180big colonies growing on TDO were picked and seededon QDO/X plates, and17of them showed positive response in the β-galactosidaseassay. After several analyses on the DDO/X plates,2pseudo-positive clones weredetected and eliminated. There were15true positive clones left.1.4Sequencing of putative positive clones and BLAST analysisThe pGADT7-specific inserts of plasmids were amplified by colony PCR to eliminateduplicates containing the same AD/Library plasmid.15plasmids isolated from positiveyeast clones were transformed into E.coli through electroporation. Using the BLASTprogram at the National Center for Biotechnology Information,7sequences from the15true positive colonies were found to have high similarity to known genes. Sevendifferent Homo sapiens genes were identified by using the yeast two-hybrid analysisusing MANF as bait for the prey of the Human Fetal brain cDNA library. RNF2is oneof them.2. Co-transformation of RNF2and MANF to verify the interaction in the yeastTo verify the interaction between MANF and RNF2in the yeast, we co-transformedGold yeast competent cell with pGADT7-RNF2and pGBKT7-MANF. It showed thatthe Gold yeast co-transformed with two plasmids grew well and turned blue on theQDO/X plates. The co-transformation assay confirmed that MANF interacted withRNF2in the yeast. 3. GST pull-down assay to test the direct binding between MANF and RNF2To test the direct binding between MANF and RNF2, we carried out a GST pull-downassay in which histidine-tagged recombinant MANF was purified on Ni-NTA agarosebeads and used as an affinity matrix to absorb purified GST-RNF2in vitro. It showedthat GST-tagged RNF2successfully bound to the histidine-tagged recombinant MANF.This result suggests that RNF2and MANF had a direct interaction.4. Immunoprecipitation assay to verify the interaction between MANF and RNF2in N2A cellsTo verify the interaction between MANF and RNF2in mammalian cells, we carriedout an immunoprecipitation assay using N2A cells stably expressing GFP-MANF. Weused anti-RNF2to immunoprecipitate soluble RNF2and its binding proteins fromlysates. GFP antibody was used to detect the co-precipitated MANF. We found thatRNF2specially interacted with MANF in N2A cells.5. RNF2and MANF interact in the nucleus5.1ER stress induces nuclear translocation of MANF and co-localization withRNF2To examine the distribution of endogenous MANF and RNF2, we performedimmunofluorescent Staining. The result showed that MANF mainly distributed in thecytosol, while RNF2localized in the nucleus in N2A cells. However, MANF wasup-regulated and translocated to nuclei where it co-localized with RNF2aftertreatment with tunicamycin (4μg/ml) for16hrs, suggesting that MANF may interactwith RNF2in the nucleus. 5.2Immunoprecipitation assay of the extraction from nuclear and cytoplasmprotein demonstrates RNF2and MANF interact in the nucleusTo investigate the exact cellular compartments of the interaction between MANF andRNF2, we isolated the fractions of nucleus and cytosol and immunoprecipitated RNF2with anti-RNF2antibody. Anti-FLAG antibody was used to detect the co-precipitatedMANF. We found that MANF specifically interacted with RNF2in nuclei.6. ER stress induces RNF2expression in neuronal cell lineIt has been reported that RNF2increased in ischemic tolerance brain and retina. Wefound that RNF2interacted with MANF. MANF is an ER stress-inducible protein.Therefore, we wonder that whether ER stress also induces RNF2expression in neuralcell line. To test it, we treated the N2A cells with ER stress inducer TM(4μg/ml)for16hrs. Western blotting showed that both RNF2and MANF could be induced by ERstress7. RNF2was up-regulated under the focal cerebral ischemia and co-localized withMANFTo investigate the pattern of RNF2expression and distribution in ischemia cerebraltissue, we established rat ischemic model with middle cerebral artery occlusion(MCAO). Double labeled immunofluorescent staining was performed at24hrs or48hrs after the reperfusion. It showed that both MANF and RNF2mainly expressed inthe neurons. RNF2expression was induced at24hrs after reperfusion and co-localizedwith MANF. However, most of the neurons lost in the cerebral cortex48hrs afterreperfusion where the expression of RNF2and MANF became weak.8. Verifying the efficacy of RNF2-siRNAThe synthetic RNF2-siRNA was transfected into N2A cells to knockdown theendogenous RNF2. After24hrs,36hrs,48hrs, and72hrs of transfection, weperformed western blotting to detect the expression of RNF2. The result showed thatthe efficacy of knockdown by RNF2-siRNA depended on the time of transfection. The most efficient time to inhibit the RNF2protein level was from36hrs to48hrs.Therefore, we choose the time point of36hrs in the subsequent experiments.9. RNF2inhibits the loss of neurons induced by ER stressTo prove the hypothesis that induction of RNF2is protective for neural cells whilethey suffer insults, we transfected RNF2cDNA or siRNA into N2A cells tooverexpress or knockdown RNF2. After treatment with TM, PI staining was used toidentify the dead neural cells. We found that RNF2-siRNA obviously increased thenumber of dead neurons, compared with control, while overexpression of RNF2reduces the PI positive cells. These findings indicate RNF2inhibited the loss ofneurons induced by ER stress.10. RNF2prevents neurons from ER stress-induced apoptosis by repressing thecleavage of caspase-3To further investigate the mechanisms of the protective effect of RNF2on neural cells,we treated N2A cells with TM and then performed western blotting to determine theexpression of cleaved caspase-3and CHOP. It showed that CHOP expression andcaspase-3cleavage were increased after TM exposure. RNF2over-expression andknockdown attenuated and aggravated caspase-3activation, respectively but notaffected the elevated level of CHOP. This result suggests that RNF2prevented neuronsfrom ER stress-induced apoptosis by repressing the cleavage of caspase-3.Conclusions:RNF2interacted with MANF both in vivo and in vitro. RNF2protected neurons fromthe insults induced by ER stress and focal cerebral ischemia.

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