α-乳白蛋白错误折叠产物诱导肿瘤细胞凋亡机制研究

发布时间：2018-03-10 12:27

本文选题：α-乳白蛋白　切入点：油酸　出处：《武汉大学》2009年博士论文　论文类型：学位论文

【摘要】：生物体内的各种功能都是通过蛋白质来行使的,而功能的行使以相应的结构为基础。因此,研究蛋白质结构与功能的关系,有助于我们了解复杂的生命现象。多种人类疾病都和蛋白质错误折叠以及聚集有关。在体内,聚集可能是蛋白质在正常或压力条件下躲过了分子伴侣和蛋白酶体的质量控制监控而造成的失控结果,也有可能执行有益的生物学功能。研究表明,不只疾病相关蛋白质,非致病蛋白质同样能够形成淀粉样纤维,而且也具有细胞毒性。α-乳白蛋白是研究蛋白质折叠和错误折叠的一个经典模型蛋白。它是溶菌酶家族的成员,虽然功能不同,但三级结构与溶菌酶十分相似。该蛋白能够与多种脂肪酸结合。去钙离子的α-乳白蛋白在一定条件下部分去折叠后,能与油酸结合,形成一种能特异性诱导肿瘤细胞凋亡的复合物HAMLET(humanα-lactalbumin made lethal to tumor cells). 我们使用荧光相图和远紫外CD相图对α-乳白蛋白酸变性的过程进行了分析。酸诱导α-乳白蛋白去折叠过程中存在两个中间态,一个是pH 3.0附近的熔球态,另外一个中间态处于pH 4.0-4.5之间。等温滴定量热实验发现α-乳白蛋白在pH 4.3条件下变性时放热明显,可能存在自身聚集现象。我们使用紫外吸收光谱等方法研究了油酸对部分去折叠的人源及牛源α-乳白蛋白的影响。结果表明,pH 4.04.5条件下油酸能够诱导α-乳白蛋白中间体形成聚集体。该聚集体在其生成的pH值环境下完全不溶,但在37℃中性条件下能够重新溶解。我们使用等温滴定量热法检测了中等酸度条件下牛源α-乳白蛋白与油酸的相互作用,得到两者的结合常数约为1×105M-1。细胞毒性实验表明,油酸诱导两种α-乳白蛋白形成的聚集体对人肺癌细胞A549及人白血病细胞HL60都具有显著的的毒性。白血病细胞对于该聚集更为敏感。以上的研究结果有助于我们进一步探索HAMLET形成的分子机制。我们使用紫外吸收光谱等方法研究了三种脂肪酸对部分去折叠的脱辅基α-乳白蛋白的影响。结果表明,对于处于pH4.0-4.5条件下的牛α-乳白蛋白折叠中间体,两种不饱和脂肪酸(油酸和亚油酸)和一种饱和脂肪酸(硬脂酸),均能够以时间依赖和浓度依赖的方式诱导其形成无定形的非纤维聚集体。该聚集体在37℃中性条件下能够重新溶解。我们使用内源荧光光谱、ANS结合、远紫外圆二色光谱以及飞行时间质谱对其进行了分析。结果表明,该聚集体在重新溶解后,结构特征类似于HAMLET/BAMLET。细胞毒性实验表明,油酸和亚油酸诱导α-乳白蛋白形成的聚集体对人肺癌细胞A549具有显著的、浓度依赖的毒性,而硬脂酸诱导的聚集则没有对该肿瘤细胞表现出明显毒性。此外,两种不饱和脂肪酸诱导α-乳白蛋白形成的聚集体能够诱导A549凋亡,因此这种聚集体有被开发成抗肿瘤药物的潜在价值。以上研究结果有助于了解脂肪酸导致的α-乳白蛋白的寡聚化现象以及HAMLET/BAMLET形成的分子机制。折叠构象的改变有可能使同一条蛋白质多肽链能够行使不同的生物学功能,深化了我们对蛋白质结构与功能关系的认识。
[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.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R341

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