p55PIK蛋白的功能预测及分析

发布时间：2018-02-16 04:35

  本文关键词： PI3K p55PIK 进化树　出处：《湖北工业大学》2017年硕士论文　论文类型：学位论文

【摘要】：磷脂酰肌醇-3-激酶(phosphatidylinositol 3 kinase,PI3K)是一种胞内磷脂酰肌醇激酶,IA型的PI3K是由调节亚基和催化亚基组成的异源二聚体。其中催化亚基有p110α、p110β、p110δ,调节亚基有p85α、p85β、p55α、p55γ(即p55PIK)、p50α。调节亚基本身没有酶活性,需要通过iSH2结构域与催化亚基p110的N端结构域结合形成稳定的复合物发挥活性。p55PIK作为PI3K的一个重要亚基,参与细胞的增殖、分化、代谢、DNA合成等途径,p55PIK与肿瘤的形成密切相关,其独特的N端结构在肿瘤细胞的发生、发展中发挥着重要的功能。现已有研究发现p55PIK有一个亚型p50PIK,这一蛋白是在p55PIK的基础上由不同的翻译起始位点翻译得到的,翻译出来的蛋白有50KD,即为p50PIK。在结构上p50PIK比p55PIK缺少了N端的氨基酸,p50PIK具体有什么样的功能仍不清楚。目的:从进化角度来解释具有独特N端结构的p55PIK的出现,是否在不同物种间高度保守,或者只是出现在高等生物中。鉴定选择性翻译起始产生的p55PIK蛋白不同亚型并对其功能进行分析,研究缺少N端氨基酸的p50PIK与p55PIK在功能上是否有区别?除了p50PIK外,p55PIK是否还有其他的亚型?若有,在结构上与p55PIK有什么区别?各自又有什么样的功能?确定p55PIK及其不同亚型在PI3K通路中如何参与各种细胞内生理活性,鉴定出与p55PIK及其亚型相互作用的不同细胞周期检查点调节蛋白,对深入研究该亚基及亚型如何调节细胞周期具有深远的意义,并可能为临床癌症的治疗提供新的方向。方法:首先用生物信息学的方法从进化的角度初步研究p55PIK的进化历史,其N端的保守结构的起源;然后通过分子克隆手段用PCR扩增,酶切,连接,转化等构建p55PIK及其亚型的相关载体;最后将构建好的载体利用转染,感染等技术转染入细胞中,用Western Blotting检测蛋白表达,用流式细胞术和Edu染色的实验手段检测细胞周期的影响,用免疫荧光的技术观察在细胞中的定位。结果:系统发育的结果显示p55PIK蛋白的N端结构在两栖,爬行类,鱼类,鸟类,哺乳动物和人类中是高度保守的,而在以果蝇和线虫为代表的低等的无脊椎动物中并没有发现;通过分子克隆技术成功构建了pLenti-p55,pLenti-p50,pLenti-p48和p3XFLAG-p55,p3XFLAG-p50,p3XFLAG-p48等载体;细胞周期检测的结果发现p55PIK能促进细胞周期,p50PIK对细胞周期没有明显的影响,同时p55PIK,p50PIK和p48PIK都定位在细胞核中。结论:系统发育树的结果揭示了p55PIK蛋白,特别是N端肽段在高等脊椎动物中可能具有特定的功能。p55PIK和p50PIK对细胞周期的不同影响,说明N24在细胞中有着特异的功能。p55PIK定位在细胞核内,可以通过p55PIK的N端结构与细胞核中的Rb,PCNA等蛋白特异性的结合来解释,而p50PIK和p48PIK是如何定位在细胞核中发挥其生物学功能的,有待进一步的研究。
[Abstract]:Phosphatidylinositol 3 kinase (PI3K) is a kind of intracellular phosphatidylinositol kinase (I3K), which is a heterodimer of phosphatidylinositol kinase type IA, which consists of regulatory subunit and catalytic subunit. The catalytic subunit of phosphatidylinositol phosphatidylinositol 3 kinasePI3K is a heterodimer composed of regulatory subunit and catalytic subunit, among which the catalytic subunit is p110 伪 -p110 尾 -p110 未, and the regulatory subunit is p85 伪 -p85 尾 -p55 伪 -p55 伪 -p55 伪 -p50 伪. The regulatory subunit itself has no enzyme activity. P55PIK is an important subunit of PI3K, which involves in cell proliferation, differentiation and metabolic synthesis. P55PIK is closely related to tumor formation through the binding of the iSH2 domain with the N-terminal domain of the catalytic subunit p110 to form a stable complex. Its unique N-terminal structure plays an important role in the development and development of tumor cells. It has been found that p55PIK has a subtype p50PIK, which is derived from the translation of different translation initiation sites on the basis of p55PIK. The structure of p50PIK is less than that of p55PIK. The function of p50PIK is not clear. Objective: to explain the emergence of p55PIK with unique N-terminal structure from an evolutionary point of view. Whether highly conserved in different species or only in higher organisms. Different subtypes of p55PIK protein produced by selective translation initiation were identified and their functions were analyzed. Is there a functional difference between p50PIK and p55PIK, which lacks N-terminal amino acids? Are there any other subtypes of p55PIK besides p50PIK? If so, what is the structural difference from p55PIK? What are their respective functions? To determine how p55PIK and its different subtypes participate in various cellular physiological activities in the PI3K pathway, and to identify different cell cycle checkpoint regulators interacting with p55PIK and its subtypes. It is of great significance to further study how the subunit and its subtype regulate cell cycle, and may provide a new direction for the treatment of clinical cancer. Methods: firstly, the evolutionary history of p55PIK was preliminarily studied from the perspective of evolution by bioinformatics. The origin of the conserved structure of the N-terminal of p55PIK and its subtype was constructed by PCR amplification, restriction endonuclease digestion, ligation and transformation. Finally, the constructed vector was transfected into the cell by transfection and infection. The expression of p55PIK protein was detected by Western Blotting, the effect of cell cycle was detected by flow cytometry and Edu staining, and the localization of p55PIK protein was observed by immunofluorescence technique. Results: phylogenetic results showed that the N-terminal structure of p55PIK protein was amphibious. Highly conserved in reptiles, fish, birds, mammals and humans, but not found in the lower invertebrates represented by Drosophila and nematodes, pLenti-p55pLenti-p50 p50 pLenti-p48 and p3XFLAG-p53XLAG-p503XFLAG-p48 were successfully constructed by molecular cloning. The results of cell cycle detection showed that p55PIK could promote cell cycle and p50PIK had no obvious effect on cell cycle, while p55PIKp50PIK and p48PIK were located in the nucleus. Conclusion: the phylogenetic tree revealed p55PIK protein. In particular, N-terminal peptides may have specific functions in higher vertebrates. P55PIK and p50PIK have different effects on cell cycle, indicating that N24 has a specific function. P55PIK is located in the nucleus. It can be explained by the specific binding of p55PIK N-terminal structure to RbPIK and other proteins in the nucleus. However, how p50PIK and p48PIK play their biological functions in the nucleus needs further study.
【学位授予单位】：湖北工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;Q51

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