本文選題：p53 + IFITM3��； 參考：《中國(guó)農(nóng)業(yè)科學(xué)院》2015年博士論文

【摘要】：日本腦炎病毒(Japanese encephalitis virus,JEV)是一種經(jīng)蚊蟲(chóng)傳播的黃病毒,能引起中樞神經(jīng)系統(tǒng)損傷和豬的繁殖障礙,具有重要的公共衛(wèi)生學(xué)意義。腫瘤抑制因子p53具有參與細(xì)胞周期捕獲、細(xì)胞代謝、誘導(dǎo)細(xì)胞凋亡和先天性免疫的作用。與野生型小鼠相比,JEV感染p53缺失小鼠能引起較高病毒血癥、死亡率,腦組織出現(xiàn)更為嚴(yán)重的炎癥反應(yīng)和病理變化。表明p53在宿主抗JEV感染中具有重要作用。除了已報(bào)道的p53增強(qiáng)I-IFN介導(dǎo)的抗病毒先天性免疫和誘導(dǎo)細(xì)胞凋亡外,是否還存在其他抗病毒先天性免疫調(diào)控機(jī)制?為了進(jìn)一步闡明p53抗JEV的分子機(jī)制及深入理解p53介導(dǎo)的細(xì)胞與JEV相互作用,遂進(jìn)行了本課題研究。JEV感染小鼠腦組織表達(dá)譜芯片發(fā)現(xiàn),JEV感染的小鼠腦組織中IFITM3 mRNA水平顯著上調(diào),p53WT小鼠IFITM3 mRNA水平高于p53KO小鼠。這種變化與p53轉(zhuǎn)錄調(diào)控的靶基因IRF9、TLR3和GBP1等具有相似性,提示兩者間存在某種聯(lián)系。p53生物學(xué)功能的發(fā)揮多依賴于其轉(zhuǎn)錄活性,然而,在分析p53不同狀態(tài)下,IFITM3 mRNA水平變化時(shí),發(fā)現(xiàn)IFITM3的轉(zhuǎn)錄并不受p53的影響,說(shuō)明兩者并沒(méi)有直接關(guān)系。但當(dāng)對(duì)IFITM3蛋白表達(dá)變化進(jìn)行檢測(cè)時(shí),發(fā)現(xiàn)IFITM3的蛋白豐度隨p53信號(hào)通路的激活而明顯上升,表明p53間接上調(diào)IFITM3的蛋白表達(dá)�？紤]到蛋白棕櫚�；揎棇�(duì)蛋白定位、穩(wěn)定性、生物學(xué)功能的重要作用,對(duì)p53上調(diào)IFITM3蛋白豐度與蛋白棕櫚�；揎椀年P(guān)系進(jìn)行了評(píng)估,發(fā)現(xiàn)棕櫚�；种苿�2-BP處理廢除了p53與IFITM3間的調(diào)控關(guān)系。IFITM3棕櫚�；揎椀囊种坪笃涞鞍妆磉_(dá)明顯下降,而轉(zhuǎn)錄水平未受影響。進(jìn)一步探究證明2-BP處理后,IFITM3蛋白穩(wěn)定性明顯下降,半衰期由5h衰減為2h,溶酶體途徑抑制劑Leupeptin處理能明顯恢復(fù)IFITM3蛋白含量,并延長(zhǎng)其半衰期至10h,說(shuō)明IFITM3降解的主要途徑為溶酶體途徑。IFIITM3棕櫚�；揎椖茉鰪�(qiáng)IFITM3蛋白穩(wěn)定性,抑制其在溶酶體中降解,也證實(shí)了p53可以間接調(diào)控IFITM3棕櫚�；揎棽⒃鰪�(qiáng)其蛋白穩(wěn)定性。蛋白棕櫚�；揎検蹹HHC蛋白和PPT的可逆調(diào)節(jié),因此,我們假設(shè)p53直接轉(zhuǎn)錄調(diào)控負(fù)責(zé)IFITM3棕櫚�；揎椀哪承┟�,如正向調(diào)控具有蛋白乙酰轉(zhuǎn)移酶活性的DHHC家族成員(負(fù)責(zé)蛋白棕櫚�；揎�),或負(fù)調(diào)控乙酰蛋白硫酯酶APTs和蛋白棕櫚酸硫酯酶PPTs(負(fù)責(zé)蛋白去棕櫚�；揎�),從而間接調(diào)節(jié)IFITM3的蛋白穩(wěn)定性。隨后對(duì)p53轉(zhuǎn)錄調(diào)控ZDHHC和PPT家族成員的能力進(jìn)行了分析,發(fā)現(xiàn)在不同細(xì)胞系中ZDHHC1 mRNA水平均隨p53狀態(tài)而改變。對(duì)ZDHHC1蛋白水平檢測(cè)也證實(shí)p53同樣可以上調(diào)IFITM3的蛋白水平,從而說(shuō)明了p53轉(zhuǎn)錄調(diào)控IFITM3表達(dá)。IFITM3蛋白棕櫚�；揎椑谄涞鞍追�(wěn)定性,ZDHHC1若修飾IFITM3,其蛋白表達(dá)和穩(wěn)定性也會(huì)增加。ZDHHC1過(guò)表達(dá)上調(diào)IFITM3蛋白表達(dá),并不影響其轉(zhuǎn)錄水平;ZDHHC1knockdown降低了IFITM3的蛋白豐度,說(shuō)明了p53調(diào)控的中間分子ZDHHC1的確利于IFITM3蛋白表達(dá)與穩(wěn)定性。進(jìn)一步試驗(yàn)確認(rèn)了ZDHHC1和IFITM3的表達(dá)變化與p53的蛋白表達(dá)變化存在一致性,證實(shí)了p53-ZDHHC1-IFITM3調(diào)控通路的存在。為闡明p53-ZDHHC1-IFITM3通路在JEV復(fù)制過(guò)程中的作用,分別對(duì)各個(gè)分子單獨(dú)或協(xié)同作用于JEV復(fù)制的影響進(jìn)行了分析。證實(shí)了p53通過(guò)轉(zhuǎn)錄調(diào)控ZDHHC1表達(dá)間接增強(qiáng)IFITM3蛋白穩(wěn)定性,從而抑制JEV復(fù)制的作用。另外,JEV也存在拮抗p53抗病毒先天性免疫的機(jī)制,NS4A蛋白可將p53扣留在細(xì)胞漿中,抑制p53入核和轉(zhuǎn)錄活性的發(fā)揮,并利于JEV的復(fù)制。本研究證實(shí)了p53-ZDHHC1-IFITM3調(diào)控通路的存在,并闡明了抑制JEV復(fù)制的生物學(xué)作用,揭示了p53抗JEV復(fù)制的新的分子機(jī)制,也說(shuō)明了p53參與抗病毒先天性免疫的多樣性與靈活性。p53調(diào)控ZDHHC1的表達(dá),將p53與蛋白棕櫚�；揎椔�(lián)系起來(lái),拓展了p53的生物學(xué)作用。p53-ZDHHC1-IFITM3通路的發(fā)現(xiàn)為進(jìn)一步研究p53抗病毒的分子機(jī)制及開(kāi)發(fā)抗病毒藥物提供了一定參考依據(jù)。
[Abstract]:Japanese encephalitis virus (JEV) is a kind of yellow virus transmitted by mosquitoes, which can cause the damage of the central nervous system and the reproductive barrier of pigs. It has important public health significance. The tumor suppressor factor p53 has the role of participating in cell cycle capture, cell metabolism, inducing cell apoptosis and innate immunity. JEV infected mice with p53 deficiency can cause hyperviremia, mortality, and more serious inflammatory and pathological changes in the brain tissue. It shows that p53 plays an important role in the host anti JEV infection. It is still existing in addition to the reported p53 enhanced I-IFN mediated antiviral congenital immunity and induction of apoptosis. In order to further elucidate the molecular mechanism of p53 anti JEV and to understand the interaction of p53 mediated cells and JEV, this subject studied the brain tissue expression profile of.JEV infected mice, and the level of IFITM3 mRNA in the brain tissues of JEV infected mice was significantly up-regulated, and IFITM3 mRNA in p53WT mice. It is higher than p53KO mice. This change is similar to the target gene IRF9, TLR3 and GBP1 of p53 transcriptional regulation, suggesting that there is a certain link between the biological function of.P53 and its transcriptional activity. However, when the mRNA level of IFITM3 is changed in different states of p53, the transcription of IFITM3 is not affected by p53, indicating two But when the expression of IFITM3 protein was detected, it was found that the protein abundance of IFITM3 increased obviously with the activation of p53 signaling pathway, indicating that p53 up-regulated the protein expression of IFITM3. Considering the role of protein palmitic modification on protein localization, stability, and biological function, p53 up regulation of IFITM3 eggs The relationship between white abundance and protein palmiylation modification was evaluated. Palmitoylation inhibitor 2-BP treatment abolished the regulatory relationship between p53 and IFITM3. The protein expression decreased markedly after the inhibition of.IFITM3 palmitic modification, while the transcriptional level was not affected. Further exploration proved that after 2-BP treatment, the stability of IFITM3 protein decreased significantly. The half-life decreased from 5h to 2h, and the lysosome pathway inhibitor Leupeptin treatment could obviously restore the IFITM3 protein content and prolong its half-life to 10h, indicating that the main pathway for the degradation of IFITM3 is that the lysosome pathway.IFIITM3 palmitic modification can enhance the stability of IFITM3 protein and inhibit its degradation in the lysosome, which also confirms that p53 can be indirectly regulated. IFITM3 palmiylation modifies and enhances its protein stability. Protein palmitylation modification is reversible by DHHC protein and PPT, so we assume that p53 direct transcriptional regulation is responsible for some enzymes of IFITM3 palmiylation modification, such as the positive regulation of DHHC family members with protein acetyltransferase activity (responsible for protein palmitic modification), or The negative regulation of acetyl protein thioesterase APTs and protein palmitate thioesterase PPTs (responsible for protein depalmiylation modification) indirectly regulates the protein stability of IFITM3. Subsequently, the ability of p53 transcriptional regulation of ZDHHC and PPT family members is analyzed. It is found that the level of ZDHHC1 mRNA in different cell lines is changed with p53 state. To ZDHHC1 eggs. The white level test also confirmed that p53 can also increase the protein level of IFITM3, thus indicating that p53 transcriptional regulation IFITM3 expression of.IFITM3 protein palmiylation is beneficial to its protein stability. If ZDHHC1 modifies IFITM3, its protein expression and stability also increase the over expression of.ZDHHC1 to up regulate the expression of IFITM3 protein, and does not affect its transcriptional level; ZDHH; ZDHH. C1knockdown reduced the protein abundance of IFITM3, indicating that the intermediate molecule ZDHHC1 regulated by p53 was indeed beneficial to the expression and stability of IFITM3 protein. Further experiments confirmed that the changes in the expression of ZDHHC1 and IFITM3 were consistent with the changes in the protein expression of p53, and confirmed the existence of p53-ZDHHC1-IFITM3 modulation pathway. In the process of JEV replication, the effect of individual or synergistic action on JEV replication is analyzed. It is confirmed that p53 can indirectly enhance the stability of IFITM3 protein by transcriptional regulation of ZDHHC1, thus inhibiting the role of JEV replication. In addition, JEV also has a mechanism to antagonize the innate immunity of p53, and NS4A protein can be used for P. 53 detain in the cytoplasm, inhibit the play of p53 nucleation and transcriptional activity, and facilitate the replication of JEV. This study confirmed the existence of p53-ZDHHC1-IFITM3 regulation pathway, clarified the biological function of inhibiting the replication of JEV, revealed the new molecular mechanism of p53 anti JEV replication, and also explained the diversity and spirit of p53 to participate in the antiviral innate immunity. The activity of.P53 regulates the expression of ZDHHC1, linking p53 with protein palmitic modification, expanding the discovery of the biological role of p53 and the discovery of the.P53-ZDHHC1-IFITM3 pathway, which provides a reference for further research on the molecular mechanism of p53 and the development of antiviral drugs.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65

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