合成细胞环境中蛋清肽膜亲和力与ACE抑制活力相关性研究

Recently, it is considered as a crucial therapy to inhibit the activity of angiotensin I-converting enzyme (ACE) by food derived peptides. However, most of the in vitro ACE-inhibitory activity determination methods are based on unbound ACE system, while studies indicated the key factor for in vivo blood pressure regulation is the ACE bound to the surface of cellular membrane. Thus, it was significantly important to investigate the relationship between membrane affinity and ACE-inhibitory activity peptides in cellular membrane environment. To solve this problem, the synthetic cellular environment was established and verified by synthetic biology and molecular biology methods, and then effects of egg white peptides with various sequence reorganized by Fmoc solid phase on membraneaffinity and ACE-inhibitory activity were investigated by atomic force microscope and liquid chromatography. The project aims to enlighten the relationship between membrane affinity and ACE-inhibitory activity of peptides in the synthetic cellular environment, and to establish the structure-activity relationship model of ACE-inhibitory peptides based on the membrane affinity. The research may provide new theoretical basis for further screening, sequence reorganization and processing application of high-activity food derived ACE-inhibitory peptides with successful establishment of structure-activity relationship model of ACE-inhibitory peptides based on the synthetic cellular environment.

利用食源性多肽抑制血管紧张素转化酶（ACE）活力是调节机体血压的重要策略。研究表明结合在细胞膜表面的ACE是生物体内血压调节的关键因子，但是目前普遍采用游离的ACE体系来研究食源性多肽的体外抑制活力，忽略了ACE在体内的膜结合环境。因此，明确多肽膜亲和力变化对膜结合ACE抑制活力的影响规律，是食源性ACE抑制肽研究中亟待解决的重要问题。本项目拟利用合成生物学和分子生物学的方法构建并验证ACE的合成细胞环境体系；以蛋清源ACE抑制肽为切入点,通过Fmoc固相合成、原子力显微镜和液相色谱研究多肽结构改造对膜亲和力和膜结合ACE抑制活力的影响，探索合成细胞环境中多肽膜亲和力与ACE抑制活力之间的相关性，建立基于膜亲和力的ACE抑制肽构效关系模型。本项目有望阐明ACE合成细胞环境体系中多肽膜亲和力与ACE抑制活力之间的相关性，为高活性食源性ACE抑制肽的筛选改造和加工应用提供新的理论依据。

为构建ACE合成细胞环境，本研究通过膜蛋白表达重构技术建立了膜结合ACE C结构域蛋白模型。利用CHO-K1细胞表达获得了ACE C结构域蛋白，其活力为0.53±0.06U/μg；酶促动力学结果表明细胞膜环境降低了ACE C结构域蛋白与底物HHL之间亲和力，同时提升了反应速度，其在游离体系和膜结合体系中Km值分别为114.48548±29.14676μM和158.44324±43.74532μM，Vmax值分别为6.42337±0.72099μM/min和10.45299±1.46687μM/min；通过对蛋清肽进行结构改造，发现部分蛋清肽的ACE抑制活性在游离体系和膜结合体系中存在显著差异，蛋清肽C末端氨基酸对其活力有重要影响；通过分子描述符对活性肽结构进行参数化后，利用支持向量机和粒子群优化算法建立了构效关系模型；利用分子动力学模拟技术探索了蛋清肽在游离和膜结合ACE C结构域蛋白体系中的作用机制，结果表明蛋清肽RVPSL在游离和膜结合ACE体系中结合能分别为-318.929±1.828kJ/mol和-355.852±2.168kJ/mol，肽和蛋白分子间主要通过静电力、氢键和疏水相互作用维持体系稳定，ACE C结构域蛋白上的Ala961、Trp962、Glu989、His992和Glu1016在两个体系中均与RVPSL存在相互作用。以上实验结果确证了细胞膜环境对ACE C结构域蛋白活力及ACE抑制肽结构偏好性的影响，为高活性ACE抑制肽的筛选改造和加工应用提供了一定实验依据。