本文選題：α-乳白蛋白　切入點(diǎn)：油酸　出處：《武漢大學(xué)》2009年博士論文　論文類型：學(xué)位論文

【摘要】：生物體內(nèi)的各種功能都是通過蛋白質(zhì)來行使的,而功能的行使以相應(yīng)的結(jié)構(gòu)為基礎(chǔ)。因此,研究蛋白質(zhì)結(jié)構(gòu)與功能的關(guān)系,有助于我們了解復(fù)雜的生命現(xiàn)象。多種人類疾病都和蛋白質(zhì)錯誤折疊以及聚集有關(guān)。在體內(nèi),聚集可能是蛋白質(zhì)在正�；驂毫l件下躲過了分子伴侶和蛋白酶體的質(zhì)量控制監(jiān)控而造成的失控結(jié)果,也有可能執(zhí)行有益的生物學(xué)功能。研究表明,不只疾病相關(guān)蛋白質(zhì),非致病蛋白質(zhì)同樣能夠形成淀粉樣纖維,而且也具有細(xì)胞毒性。α-乳白蛋白是研究蛋白質(zhì)折疊和錯誤折疊的一個經(jīng)典模型蛋白。它是溶菌酶家族的成員,雖然功能不同,但三級結(jié)構(gòu)與溶菌酶十分相似。該蛋白能夠與多種脂肪酸結(jié)合。去鈣離子的α-乳白蛋白在一定條件下部分去折疊后,能與油酸結(jié)合,形成一種能特異性誘導(dǎo)腫瘤細(xì)胞凋亡的復(fù)合物HAMLET(humanα-lactalbumin made lethal to tumor cells). 我們使用熒光相圖和遠(yuǎn)紫外CD相圖對α-乳白蛋白酸變性的過程進(jìn)行了分析。酸誘導(dǎo)α-乳白蛋白去折疊過程中存在兩個中間態(tài),一個是pH 3.0附近的熔球態(tài),另外一個中間態(tài)處于pH 4.0-4.5之間。等溫滴定量熱實(shí)驗(yàn)發(fā)現(xiàn)α-乳白蛋白在pH 4.3條件下變性時(shí)放熱明顯,可能存在自身聚集現(xiàn)象。我們使用紫外吸收光譜等方法研究了油酸對部分去折疊的人源及牛源α-乳白蛋白的影響。結(jié)果表明,pH 4.04.5條件下油酸能夠誘導(dǎo)α-乳白蛋白中間體形成聚集體。該聚集體在其生成的pH值環(huán)境下完全不溶,但在37℃中性條件下能夠重新溶解。我們使用等溫滴定量熱法檢測了中等酸度條件下牛源α-乳白蛋白與油酸的相互作用,得到兩者的結(jié)合常數(shù)約為1×105M-1。細(xì)胞毒性實(shí)驗(yàn)表明,油酸誘導(dǎo)兩種α-乳白蛋白形成的聚集體對人肺癌細(xì)胞A549及人白血病細(xì)胞HL60都具有顯著的的毒性。白血病細(xì)胞對于該聚集更為敏感。以上的研究結(jié)果有助于我們進(jìn)一步探索HAMLET形成的分子機(jī)制。 我們使用紫外吸收光譜等方法研究了三種脂肪酸對部分去折疊的脫輔基α-乳白蛋白的影響。結(jié)果表明,對于處于pH4.0-4.5條件下的牛α-乳白蛋白折疊中間體,兩種不飽和脂肪酸(油酸和亞油酸)和一種飽和脂肪酸(硬脂酸),均能夠以時(shí)間依賴和濃度依賴的方式誘導(dǎo)其形成無定形的非纖維聚集體。該聚集體在37℃中性條件下能夠重新溶解。我們使用內(nèi)源熒光光譜、ANS結(jié)合、遠(yuǎn)紫外圓二色光譜以及飛行時(shí)間質(zhì)譜對其進(jìn)行了分析。結(jié)果表明,該聚集體在重新溶解后,結(jié)構(gòu)特征類似于HAMLET/BAMLET。細(xì)胞毒性實(shí)驗(yàn)表明,油酸和亞油酸誘導(dǎo)α-乳白蛋白形成的聚集體對人肺癌細(xì)胞A549具有顯著的、濃度依賴的毒性,而硬脂酸誘導(dǎo)的聚集則沒有對該腫瘤細(xì)胞表現(xiàn)出明顯毒性。此外,兩種不飽和脂肪酸誘導(dǎo)α-乳白蛋白形成的聚集體能夠誘導(dǎo)A549凋亡,因此這種聚集體有被開發(fā)成抗腫瘤藥物的潛在價(jià)值。以上研究結(jié)果有助于了解脂肪酸導(dǎo)致的α-乳白蛋白的寡聚化現(xiàn)象以及HAMLET/BAMLET形成的分子機(jī)制。折疊構(gòu)象的改變有可能使同一條蛋白質(zhì)多肽鏈能夠行使不同的生物學(xué)功能,深化了我們對蛋白質(zhì)結(jié)構(gòu)與功能關(guān)系的認(rèn)識。
[Abstract]:The various functions of the organism are exercised by the protein, and the function of the corresponding structure as the foundation. Therefore, the relationship between protein structure and function research, contribute to our understanding of the complex phenomena of life. A variety of human diseases and protein misfolding and aggregation. In vivo, protein aggregation is likely to be in normal or under pressure from the quality control monitoring of molecular chaperones and proteasome caused by out of control results, it may be possible to perform useful biological functions. The results show that not only the disease related protein, non pathogenic proteins can form amyloid fibrils, but also has cytotoxicity. Alpha lactalbumin is a classic the model of protein folding and protein misfolding. It is a member of the family of lysozyme, although the function is different, but the three level structure of lysozyme and the protein are very similar. It can bind to a variety of fatty acids. The Ca2 + - deleted alpha lactalbumin can be partially folded with some oleic acid to form a HAMLET (human alpha -lactalbumin made lethal to tumor cells), which can specifically induce apoptosis of tumor cells.
We use fluorescence phase diagram and far UV CD phase diagram of alpha lactalbumin acid denaturation were analyzed. There are two intermediate acid induced by alpha lactalbumin unfolding process, a molten globule state near pH 3, in addition to an intermediate state in the pH 4.0-4.5. Isothermal titration calorimetry the experiment found that exothermic degeneration of alpha lactalbumin under the condition of pH 4.3, there may be a self aggregation phenomenon. We use UV absorption spectroscopy and other methods to study the effects of oleic acid on the unfolding of human and bovine alpha lactalbumin part. The results show that the pH 4.04.5 under the condition of oleic acid to alpha lactalbumin intermediate figure. The aggregation induced aggregates in its generated pH value environment completely insoluble, but in the condition of 37 DEG under neutral can be re dissolved. We used isothermal titration calorimetry to detect bovine lactalbumin and oil source alpha under moderate acidity conditions The interaction of acid, the binding constant is about 1 * 105M-1. showed cytotoxicity induced by oleic acid, two alpha lactalbumin aggregates have significant toxicity to A549 cells and human leukemia cell HL60 of human lung cancer. For the aggregation of leukemia cells is more sensitive. The above results help we further explore the molecular mechanism of HAMLET formation.
We use the UV absorption spectra of three kinds of fatty acids on the part of the unfolding and effects of cofactor alpha lactalbumin. The results showed that under the condition of pH4.0-4.5 in bovine - lactalbumin folding intermediates, two kinds of unsaturated fatty acids (oleic acid and linoleic acid) and a saturated fatty acid (stearic acid), can be in a time-dependent and concentration dependent manner to induce the formation of non fibrous aggregates. The amorphous aggregates can be re dissolved in the condition of 37 DEG neutral. We use fluorescence spectroscopy, ANS binding, far UV circular two light spectrum and time-of-flight mass spectrometry analyzed. Results show that the aggregates in re dissolved, the structural features similar to HAMLET/BAMLET. cell toxicity test showed that oleic acid and linoleic acid induced by alpha lactalbumin aggregates significantly on A549 human lung cancer cells, concentration dependent The toxicity of stearic acid induced aggregation is not obvious toxicity to the tumor cells. In addition, two kinds of unsaturated fatty acids induced by alpha lactalbumin aggregates can induce apoptosis of A549, so it has been developed into aggregates potential antitumor drugs. The research results are helpful to understand the fat acid to alpha lactalbumin oligomerization and molecular mechanism of HAMLET/BAMLET formation. The folding conformation changes are likely to make the same protein polypeptide can perform different biological functions, deepen our understanding of the relationship between protein structure and function.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R341

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本文編號：1593334