乙酰化修饰在二型干扰素受体介导的信号通路中作用的研究和溶瘤腺病毒对PLC细胞作用机制的研究

发布时间：2018-05-03 00:31

本文选题：干扰素gamma受体 + STAT1　；参考：《浙江理工大学》2011年硕士论文

【摘要】：蛋白质的转录翻译后修饰在生命活动中扮演着重要的角色，如磷酸化、乙酰化、甲基化和泛素化等。乙酰化可以调节组蛋白和非组蛋白的活性。最近又有文章报道乙酰化可以调节代谢循环中相关酶的活性。现在乙酰化修饰已经成为了研究的热点。虽然已经发现了很多蛋白质的乙酰化修饰，但有关二型干扰素受体（IFNgR）的乙酰化修饰的研究却很少。本课题主要从两方面研究乙酰化在IFNgR介导的信号通路中的作用。一方面，我们研究IFNgR是否会发生乙酰化以及乙酰化对其功能的影响。另一方面，我们研究STAT1新的乙酰化位点以及这些新位点对其功能的影响。 IFNgR乙酰化修饰方面，我们构建了能够表达IFNgR1Full Lengt（hIFNgR1FL）、IFNgR1Cytoplasmic Domain（IFNgR1CD）、IFNgR2FL和IFNgR2CD的质粒，同时也构建了一系列IFNgR1CD的点突变体和截断体。然后经IP和Western blot等方法检测发现：在CBP的作用下IFNgR1FL、IFNgR1CD和IFNgR2FL会发生明显的乙酰化修饰，而IFNgR2CD基本检测不到乙酰化修饰。我们已经送出乙酰化IFNgR1FL和IFNgR2FL的蛋白样品，用于质谱分析。对于IFNgR1CD突变体的实验发现，K~(271,272)、K~(303)、K~(459)和K~(473)是IFNgR1CD发生乙酰化的关键位点。 STAT1乙酰化修饰方面，我们用IP和Western blot实验发现内源的STAT1在IFN-γ的诱导下会发生乙酰化；质谱的结果显示我们发现了STAT1新的乙酰化位点（K~(193)和K~(697)），STAT1的K193R和K697R突变体的实验结果进一步证实了这两个位点是STAT1发生乙酰化的关键位点；Western blot和荧光素酶报告基因检测显示K~(193)和K~(697)的乙酰化修饰不影响STAT1同源二聚体的形成并且对STAT1的转录活性的影响不如Y701那样显著。本课题还将进一步深入研究，希望为乙酰化修饰的研究出一份力。溶瘤腺病毒Ad.sp-E1A(△24)-IL-24对人肝癌细胞株PLC的作用机制尚不清楚。采用流式细胞仪检测PLC经PBS、Ad.sp-E1A(△24)以及Ad.sp-E1A(△24)-IL-24处理48h后细胞周期的变化；利用Western blot检测PLC经PBS、Ad.sp-E1A(△24)以及Ad.sp-E1A(△24)-IL-24处理24h、48h后细胞中凋亡信号相关蛋白表达量的变化。实验结果表明：Ad.sp-E1A(△24)和Ad.sp-E1A(△24)-IL-24能将PLC细胞周期阻滞在S期，S期比值分别为(43.74±6.61)%，(49.48±7.60)%，两者与未感染病毒的对照组(18.91±1.83)%进行统计学差异分析，P值均0.05；同时，Western blot检测发现随着处理时间的增加，病毒处理组中CHOP的表达量，Caspase12的剪切水平及STAT3、p38MAPK的磷酸化水平都有明显提高，尤其以Ad.sp-E1A(△24)-IL-24处理组相对较为明显，，SOCS3的表达量病毒处理组与对照组均没有显著差异。上述结果表明：溶瘤腺病毒Ad.sp-E1A(△24)-IL-24能有效阻滞PLC细胞周期在S期，并可能通过内质网压力、STAT3与MAPK信号通路等多种途径诱发PLC细胞凋亡。
[Abstract]:Posttranslational modification of proteins plays an important role in life activities, such as phosphorylation, acetylation, methylation and ubiquitin. Acetylation regulates the activity of histone and non-histone. Recently, it has been reported that acetylation can regulate the activity of enzymes in metabolic cycle. Now acetylation modification has become a hot topic. Although many protein acetylation modifications have been found, few studies have been conducted on the acetylation modification of IFNgR- type 2 interferon receptor (IFNgR). This study focuses on the role of acetylation in IFNgR-mediated signaling pathways. On the one hand, we study whether acetylation occurs in IFNgR and the effect of acetylation on its function. On the other hand, we study the new acetylation sites of STAT1 and their effects on its function. In the aspect of IFNgR acetylation modification, we constructed a series of IFNgR1CD point mutants and truncates, which can express IFNgR1Full engtr hIFNgR1FL1, IFNgR1Cytoplasmic domain, IFNgR1CDFL and IFNgR2CD. Then by IP and Western blot, it was found that IFNgR1FLFL-IFNgR1CD and IFNgR2FL had obvious acetylation modification under the action of CBP, but IFNgR2CD could not detect acetylation modification. We have sent out protein samples of acetylated IFNgR1FL and IFNgR2FL for mass spectrometry. For the IFNgR1CD mutants, it was found that the key sites for acetylation of IFNgR1CD were KG271272KC303KH459 and KP473). In the aspect of STAT1 acetylation modification, we used IP and Western blot experiments to find that endogenous STAT1 would be acetylated under the induction of IFN- 纬. The results of mass spectrometry show that we have found a new acetylated site of STAT1 and the K193R and K697R mutants of KG697T1and K697R, which further confirm that these two sites are the key sites for acetylation of STAT1. Western blot and luciferase reporter gene detection. The results showed that the acetylation modification of STAT1 and KG697) did not affect the formation of STAT1 homopolymer and the effect on the transcriptional activity of STAT1 was not as significant as that of Y701. This subject will be further studied, hoping to provide a contribution to the study of acetylation modification. The mechanism of adenolytic adenovirus Ad.sp-E1A (24)-IL-24) on human hepatoma cell line PLC is unclear. Flow cytometry was used to detect the cell cycle changes of PLC treated with 24)-IL-24 and Ad.sp-E1A (24) and Ad.sp-E1A (24)-IL-24) for 48h, and Western blot was used to detect the expression of apoptosis-related proteins in PLC treated with PLC for 24 h and Ad.sp-E1A (24)-IL-24) for 48 h. The results showed that the ratio of 24)-IL-24 to Ad.sp-E1A (24) and Ad.sp-E1A (24)-IL-24) was 43.74 卤6.61% and 49.48 卤7.60%, respectively. The expression of Caspase12 and phosphorylation of STAT3 p38 MAPK were significantly increased in virus treated group, especially in Ad.sp-E1A (24)-IL-24 treatment group). There was no significant difference between virus treatment group and control group. These results suggest that Ad.sp-E1A (24)-IL-24) can effectively block the cell cycle of PLC in S phase and induce apoptosis of PLC cells through various pathways such as endoplasmic reticulum pressure STAT3 and MAPK signaling pathway.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R3411

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