發(fā)布時(shí)間：2018-02-16 04:35

  本文關(guān)鍵詞： PI3K p55PIK 進(jìn)化樹　出處：《湖北工業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：磷脂酰肌醇-3-激酶(phosphatidylinositol 3 kinase,PI3K)是一種胞內(nèi)磷脂酰肌醇激酶,IA型的PI3K是由調(diào)節(jié)亞基和催化亞基組成的異源二聚體。其中催化亞基有p110α、p110β、p110δ,調(diào)節(jié)亞基有p85α、p85β、p55α、p55γ(即p55PIK)、p50α。調(diào)節(jié)亞基本身沒有酶活性,需要通過iSH2結(jié)構(gòu)域與催化亞基p110的N端結(jié)構(gòu)域結(jié)合形成穩(wěn)定的復(fù)合物發(fā)揮活性。p55PIK作為PI3K的一個(gè)重要亞基,參與細(xì)胞的增殖、分化、代謝、DNA合成等途徑,p55PIK與腫瘤的形成密切相關(guān),其獨(dú)特的N端結(jié)構(gòu)在腫瘤細(xì)胞的發(fā)生、發(fā)展中發(fā)揮著重要的功能�，F(xiàn)已有研究發(fā)現(xiàn)p55PIK有一個(gè)亞型p50PIK,這一蛋白是在p55PIK的基礎(chǔ)上由不同的翻譯起始位點(diǎn)翻譯得到的,翻譯出來的蛋白有50KD,即為p50PIK。在結(jié)構(gòu)上p50PIK比p55PIK缺少了N端的氨基酸,p50PIK具體有什么樣的功能仍不清楚。目的:從進(jìn)化角度來解釋具有獨(dú)特N端結(jié)構(gòu)的p55PIK的出現(xiàn),是否在不同物種間高度保守,或者只是出現(xiàn)在高等生物中。鑒定選擇性翻譯起始產(chǎn)生的p55PIK蛋白不同亞型并對(duì)其功能進(jìn)行分析,研究缺少N端氨基酸的p50PIK與p55PIK在功能上是否有區(qū)別?除了p50PIK外,p55PIK是否還有其他的亞型?若有,在結(jié)構(gòu)上與p55PIK有什么區(qū)別?各自又有什么樣的功能?確定p55PIK及其不同亞型在PI3K通路中如何參與各種細(xì)胞內(nèi)生理活性,鑒定出與p55PIK及其亞型相互作用的不同細(xì)胞周期檢查點(diǎn)調(diào)節(jié)蛋白,對(duì)深入研究該亞基及亞型如何調(diào)節(jié)細(xì)胞周期具有深遠(yuǎn)的意義,并可能為臨床癌癥的治療提供新的方向。方法:首先用生物信息學(xué)的方法從進(jìn)化的角度初步研究p55PIK的進(jìn)化歷史,其N端的保守結(jié)構(gòu)的起源;然后通過分子克隆手段用PCR擴(kuò)增,酶切,連接,轉(zhuǎn)化等構(gòu)建p55PIK及其亞型的相關(guān)載體;最后將構(gòu)建好的載體利用轉(zhuǎn)染,感染等技術(shù)轉(zhuǎn)染入細(xì)胞中,用Western Blotting檢測(cè)蛋白表達(dá),用流式細(xì)胞術(shù)和Edu染色的實(shí)驗(yàn)手段檢測(cè)細(xì)胞周期的影響,用免疫熒光的技術(shù)觀察在細(xì)胞中的定位。結(jié)果:系統(tǒng)發(fā)育的結(jié)果顯示p55PIK蛋白的N端結(jié)構(gòu)在兩棲,爬行類,魚類,鳥類,哺乳動(dòng)物和人類中是高度保守的,而在以果蠅和線蟲為代表的低等的無脊椎動(dòng)物中并沒有發(fā)現(xiàn);通過分子克隆技術(shù)成功構(gòu)建了pLenti-p55,pLenti-p50,pLenti-p48和p3XFLAG-p55,p3XFLAG-p50,p3XFLAG-p48等載體;細(xì)胞周期檢測(cè)的結(jié)果發(fā)現(xiàn)p55PIK能促進(jìn)細(xì)胞周期,p50PIK對(duì)細(xì)胞周期沒有明顯的影響,同時(shí)p55PIK,p50PIK和p48PIK都定位在細(xì)胞核中。結(jié)論:系統(tǒng)發(fā)育樹的結(jié)果揭示了p55PIK蛋白,特別是N端肽段在高等脊椎動(dòng)物中可能具有特定的功能。p55PIK和p50PIK對(duì)細(xì)胞周期的不同影響,說明N24在細(xì)胞中有著特異的功能。p55PIK定位在細(xì)胞核內(nèi),可以通過p55PIK的N端結(jié)構(gòu)與細(xì)胞核中的Rb,PCNA等蛋白特異性的結(jié)合來解釋,而p50PIK和p48PIK是如何定位在細(xì)胞核中發(fā)揮其生物學(xué)功能的,有待進(jìn)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 偽 -p110 尾 -p110 未, and the regulatory subunit is p85 偽 -p85 尾 -p55 偽 -p55 偽 -p55 偽 -p50 偽. 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.
【學(xué)位授予單位】：湖北工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78;Q51

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本文編號(hào)：1514671