磷酸化Hsp27與STAT3結(jié)合促進(jìn)自噬抑制肝細(xì)胞脂質(zhì)變性的分子機(jī)制研究
發(fā)布時(shí)間：2018-07-23 12:55
【摘要】：非酒精性脂肪肝病是指以無過量飲酒史,以肝細(xì)胞脂質(zhì)變性為特征的臨床綜合征,是常見的慢性肝病,也是導(dǎo)致終末期肝病的危險(xiǎn)因素。目前,尚無有效防治藥物。自噬是真核細(xì)胞中普遍存在的溶酶體降解程序。研究表明,自噬能夠促進(jìn)脂質(zhì)代謝,緩解肝臟脂質(zhì)變性。熱休克蛋白27(heat shock protein 27, Hsp27)是重要的分子伴侶,能夠發(fā)生磷酸化修飾,而磷酸化導(dǎo)致Hsp27聚合度和功能的改變。研究表明,Hsp27參與自噬調(diào)控。但是,Hsp27,尤其是磷酸化Hsp27參與自噬調(diào)控的具體機(jī)制尚不明確,有待進(jìn)一步研究。在本論文中,我們研究了磷酸化Hsp27在激活自噬以及抑制肝細(xì)胞脂質(zhì)變性中的作用。我們發(fā)現(xiàn)Hsp27與STAT3(信號(hào)轉(zhuǎn)導(dǎo)與轉(zhuǎn)錄激活因子3)結(jié)合,促進(jìn)自噬及脂質(zhì)代謝。首先,通過高脂喂養(yǎng)C57/BL6小鼠,建立小鼠肝臟脂質(zhì)變性模型,我們發(fā)現(xiàn)高脂喂養(yǎng)上調(diào)小鼠肝臟組織中Hsp25(人Hsp27在嚙齒類中的同源蛋白)的磷酸化和自噬水平。同樣,在體外培養(yǎng)的人肝臟L02細(xì)胞中,軟脂酸促進(jìn)Hsp27磷酸化和自噬激活。在軟脂酸處理前,利用RNA干擾下調(diào)Hsp27的表達(dá),或者利用Hsp27磷酸化抑制劑KRIBB3處理,或者過表達(dá)非磷酸化突變的Hsp27-3A,則抑制軟脂酸激活的自噬,并加劇脂質(zhì)變性。若過表達(dá)野生型的Hsp27-WT或者持續(xù)磷酸化突變的Hsp27-3D,則提高L02細(xì)胞的自噬水平,并緩解脂質(zhì)變性。利用羥氯喹抑制自噬體的降解,則阻斷磷酸化的Hsp27對(duì)脂質(zhì)變性的緩解作用。在高脂喂養(yǎng)的小鼠模型中,利用KRIBB3抑制Hsp25磷酸化,則抑制自噬激活,并加劇肝臟脂質(zhì)變性。我們進(jìn)一步研究了Hsp27磷酸化激活自噬的分子機(jī)制。通過免疫共沉淀實(shí)驗(yàn),我們發(fā)現(xiàn)Hsp27能與STAT3形成復(fù)合物,更為重要的是,軟脂酸處理后,Hsp27與STAT3形成的復(fù)合物增加,與此同時(shí),STAT3與PKR的復(fù)合物明顯減少。如果利用RNA干擾下調(diào)Hsp27的表達(dá),則抑制STAT3從PKR解離。如果利用KRIBB3抑制Hsp27磷酸化,則抑制Hsp27/STAT3復(fù)合體形成,并抑制STAT3從PKR解離。此外,過表達(dá)野生型的Hsp27-WT和持續(xù)磷酸化突變的Hsp27-3D,則促進(jìn)STAT3從PKR解離。過表達(dá)非磷酸化突變的Hsp27-3A,則抑制STAT3從PKR的解離。與野生型的Hsp27-WT相比,持續(xù)磷酸化突變的Hsp27-3D與STAT3結(jié)合的能力更強(qiáng)。綜上所述,本論文的研究發(fā)現(xiàn),磷酸化Hsp27與STAT3結(jié)合,干擾了STAT3與PKR復(fù)合物的形成,削弱STAT3對(duì)PKR的抑制作用,促進(jìn)PKR磷酸化eIF2a,激活自噬,從而緩解脂質(zhì)變性。本論文的研究結(jié)果明確了磷酸化Hsp27在自噬活化中的重要作用,深入揭示了磷酸化Hsp27的功能。同時(shí),揭示了磷酸化Hsp27活化自噬,促進(jìn)脂質(zhì)分解的作用,為臨床采用促進(jìn)脂質(zhì)代謝防治脂質(zhì)變性提供理論基礎(chǔ)。
[Abstract]:Non-alcoholic fatty liver disease is a clinical syndrome characterized by liver cell lipids degeneration without excessive drinking history. It is a common chronic liver disease and a risk factor leading to end-stage liver disease. At present, there is no effective control drug. Autophagy is a common lysosomal degradation process in eukaryotic cells. Studies have shown that autophagy can promote lipid metabolism and alleviate liver lipid degeneration. Heat shock protein 27 (Hsp27) is an important molecular chaperone capable of phosphorylation modification, while phosphorylation changes the degree of polymerization and function of Hsp27. The results showed that Hsp27 was involved in autophagy regulation. However, the mechanism of Hsp27, especially phosphorylated Hsp27, involved in autophagy regulation is not clear, which needs further study. In this study, we investigated the role of phosphorylated Hsp27 in activating autophagy and inhibiting hepatocyte lipid degeneration. We found that Hsp27 binds to STAT3 (signal transduction and transcription activator 3) and promotes autophagy and lipid metabolism. Firstly, the lipids denaturation model was established in C57/BL6 mice by high-fat feeding. We found that high-fat feeding up-regulated the phosphorylation and autophagy level of Hsp25 (homologous protein of human Hsp27 in rodents) in mice liver. Similarly, in cultured human liver L02 cells, palmitate promotes Hsp27 phosphorylation and autophagy activation. Before palmitic acid treatment, Hsp27 expression was down-regulated by RNA interference, or treated with Hsp27 phosphorylation inhibitor KRIBB3 or overexpression of non-phosphorylated mutant Hsp27-3A, which inhibited palmitic acid-activated autophagy and aggravated lipid denaturation. Overexpression of wild-type Hsp27-WT or continuous phosphorylation mutation Hsp27-3D increased autophagy level of L02 cells and alleviated lipid denaturation. Hydroxychloroquine was used to inhibit the degradation of autophagy and to block the effect of phosphorylated Hsp27 on lipid denaturation. In hyperlipidemic mice, KRIBB3 inhibited Hsp25 phosphorylation, inhibited autophagy activation and aggravated liver lipid degeneration. We further studied the molecular mechanism of Hsp27 phosphorylation activated autophagy. By immunoprecipitation, we found that Hsp27 can form complex with STAT3, and more importantly, the complex of Hsp27 and STAT3 increased after palmitate treatment, while the complex of statin and PKR decreased significantly. If the expression of Hsp27 was down-regulated by RNA interference, the dissociation of STAT3 from PKR was inhibited. If KRIBB3 was used to inhibit the phosphorylation of Hsp27, the formation of Hsp27/STAT3 complex and the dissociation of STAT3 from PKR were inhibited. In addition, overexpression of wild-type Hsp27-WT and persistent phosphorylation mutation Hsp27-3D promoted the dissociation of STAT3 from PKR. Overexpression of non-phosphorylated Hsp27-3 A inhibits the dissociation of STAT3 from PKR. Compared with wild type Hsp27-WT, Hsp27-3D with persistent phosphorylation mutation has stronger ability to bind to STAT3. In conclusion, we found that the combination of phosphorylated Hsp27 and STAT3 interferes with the formation of STAT3 / PKR complex, weakens the inhibitory effect of STAT3 on PKR, promotes PKR phosphorylation of eIF2a, activates autophagy, and alleviates lipid denaturation. In this paper, the important role of phosphorylated Hsp27 in autophagy activation was clarified, and the function of phosphorylated Hsp27 was revealed. At the same time, it revealed the role of phosphorylated Hsp27 activated autophagy and promoted lipid decomposition, which provided a theoretical basis for the prevention and treatment of lipid denaturation by promoting lipid metabolism.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R575
本文編號(hào)：2139479
[Abstract]:Non-alcoholic fatty liver disease is a clinical syndrome characterized by liver cell lipids degeneration without excessive drinking history. It is a common chronic liver disease and a risk factor leading to end-stage liver disease. At present, there is no effective control drug. Autophagy is a common lysosomal degradation process in eukaryotic cells. Studies have shown that autophagy can promote lipid metabolism and alleviate liver lipid degeneration. Heat shock protein 27 (Hsp27) is an important molecular chaperone capable of phosphorylation modification, while phosphorylation changes the degree of polymerization and function of Hsp27. The results showed that Hsp27 was involved in autophagy regulation. However, the mechanism of Hsp27, especially phosphorylated Hsp27, involved in autophagy regulation is not clear, which needs further study. In this study, we investigated the role of phosphorylated Hsp27 in activating autophagy and inhibiting hepatocyte lipid degeneration. We found that Hsp27 binds to STAT3 (signal transduction and transcription activator 3) and promotes autophagy and lipid metabolism. Firstly, the lipids denaturation model was established in C57/BL6 mice by high-fat feeding. We found that high-fat feeding up-regulated the phosphorylation and autophagy level of Hsp25 (homologous protein of human Hsp27 in rodents) in mice liver. Similarly, in cultured human liver L02 cells, palmitate promotes Hsp27 phosphorylation and autophagy activation. Before palmitic acid treatment, Hsp27 expression was down-regulated by RNA interference, or treated with Hsp27 phosphorylation inhibitor KRIBB3 or overexpression of non-phosphorylated mutant Hsp27-3A, which inhibited palmitic acid-activated autophagy and aggravated lipid denaturation. Overexpression of wild-type Hsp27-WT or continuous phosphorylation mutation Hsp27-3D increased autophagy level of L02 cells and alleviated lipid denaturation. Hydroxychloroquine was used to inhibit the degradation of autophagy and to block the effect of phosphorylated Hsp27 on lipid denaturation. In hyperlipidemic mice, KRIBB3 inhibited Hsp25 phosphorylation, inhibited autophagy activation and aggravated liver lipid degeneration. We further studied the molecular mechanism of Hsp27 phosphorylation activated autophagy. By immunoprecipitation, we found that Hsp27 can form complex with STAT3, and more importantly, the complex of Hsp27 and STAT3 increased after palmitate treatment, while the complex of statin and PKR decreased significantly. If the expression of Hsp27 was down-regulated by RNA interference, the dissociation of STAT3 from PKR was inhibited. If KRIBB3 was used to inhibit the phosphorylation of Hsp27, the formation of Hsp27/STAT3 complex and the dissociation of STAT3 from PKR were inhibited. In addition, overexpression of wild-type Hsp27-WT and persistent phosphorylation mutation Hsp27-3D promoted the dissociation of STAT3 from PKR. Overexpression of non-phosphorylated Hsp27-3 A inhibits the dissociation of STAT3 from PKR. Compared with wild type Hsp27-WT, Hsp27-3D with persistent phosphorylation mutation has stronger ability to bind to STAT3. In conclusion, we found that the combination of phosphorylated Hsp27 and STAT3 interferes with the formation of STAT3 / PKR complex, weakens the inhibitory effect of STAT3 on PKR, promotes PKR phosphorylation of eIF2a, activates autophagy, and alleviates lipid denaturation. In this paper, the important role of phosphorylated Hsp27 in autophagy activation was clarified, and the function of phosphorylated Hsp27 was revealed. At the same time, it revealed the role of phosphorylated Hsp27 activated autophagy and promoted lipid decomposition, which provided a theoretical basis for the prevention and treatment of lipid denaturation by promoting lipid metabolism.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R575
【相似文獻(xiàn)】
相關(guān)博士學(xué)位論文 前1條
1 沈磊;磷酸化Hsp27與STAT3結(jié)合促進(jìn)自噬抑制肝細(xì)胞脂質(zhì)變性的分子機(jī)制研究[D];南京師范大學(xué);2016年
，本文編號(hào)：2139479
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