基于对p53蛋白影响及靶点构效关系的鹰嘴豆肽CPe-III抑制乳腺癌作用机制研究

Based on the domestic and foreign studies on the bioactive peptides, especially on the demand for the basic research on the rich chickpea resources in our country, as well as the research results of earlier stages, using breast cancer as the research object and p53 protein the target spot, this project characterizes the advanced structure and conformation of CPe-III through the high-resolution MS, NMR, and molecular dynamics calculation, investigates the interaction of chickpea peptide CPe-III with p53 protein and the resulted protein activity, intracellular distribution and transcriptional activity of p53 protein via molecular docking, molecular dynamics calculation, molecular biology technology and method. We will also explore the structure-activity relationships of oligopeptides, the metabolic end product of CPe-III, investigate the CPe-III's antioxidant and the effect of inducing breast cancer cell apoptosis through the newest chemistry imitation, intracellular antioxidant assessment system and apoptosis theory as well. Thereby, the antitumor mechanism of CPe-III on the cell and molecular level will be revealed.The results will provide the theoretical foundation and science basis for comprehensive and further utilization and even the development of the third generation functional food of chickpea resources, aslo provide the referential experience and thoughts for the research of structure-activity relationships of the plant peptide.

基于国内外天然产物生物活性肽的研究现状，特别是我国丰富的鹰嘴豆资源研究的需要，在前期研究基础上，本课题以乳腺癌作为研究对象，p53蛋白为作用靶点，采用高分辨率质谱、核磁共振等分析方法及分子动力学模拟表征鹰嘴豆肽CPe-III的微观构象，利用分子对接、分子动力学模拟、分子生物学技术与方法解析CPe-III与乳腺癌细胞p53蛋白间相互作用及其对p53蛋白活性、细胞内分布、转录活性的影响。并探究Cpe-III体内代谢终产物中寡肽的构效关系。同时，以最新化学模拟、细胞内抗氧化评价体系、细胞凋亡理论评价CPe-III抗氧化、诱导乳腺癌细胞凋亡的生物学功效。从而从细胞和分子水平探明CPe-III抑制乳腺癌活性的作用机制，为提高乳腺癌的治疗效果提供分子基础。研究结果可为鹰嘴豆资源的综合深度利用、将鹰嘴豆肽开发成第三代功能性食品提供理论基础和科学依据。并为植物源多肽构效关系研究提供新的思路与借鉴。

基于国内外天然产物生物活性肽的研究现状，特别是我国丰富的鹰嘴豆资源研究的需要，本课题在前期研究基础上，利用体内外实验评价鹰嘴豆粗品肽及级分CPe-III抗氧化和抑制肿瘤生物活性；采用液质联用、傅里叶-拉曼光谱、傅里叶-红外光谱分析、分子模拟等技术表征、预测CPe-III的结构及理化性质；化学合成与CPe-III具有相同组成的肽CPe-Ⅲ-S，结合体外模拟、小鼠体内实验解析其消化代谢产物；MTT法评价CPe-Ⅲ-S抑制乳腺癌细胞增殖作用，West blotting分析其上调p53蛋白表达的作用；以p53蛋白为靶点，利用分子模拟及分子动力学理论，解析CPe-III 与野生及突变p53蛋白复合物相互作用模式及其与p53 蛋白结构域的结合位点及作用，揭示了CPe-III抑制乳腺癌生物学功效的作用机理。为鹰嘴豆肽的功能开发和综合深度利用提供科学依据和理论基础，为其他植物源活性肽的结构研究特别是高级结构研究提供可借鉴的方法。