Quantitative structure-activity relationship study of food protein-derived calmodulin-binding peptides

我的实验室以前的工作已经证实了通过酶水解豌豆和亚麻籽蛋白生产含有低分子量（<1 kDa）钙调素（CaM）结合肽的蛋白水解物的可行性。由于它们的体积小，肽有很高的潜力被完整地吸收到血液循环系统中；体外试验证实对胃肠道消化酶有抗性蛋白质水解物被证明可以抑制cam依赖性代谢酶的活性，如蛋白激酶II （CaMKII）和一氧化氮合酶（NOS），这些酶与慢性疾病的发病机制有关。拟议的研究将使用蛋白质水解物作为原料，分离和鉴定有效的cam结合肽的结构特征。多肽将使用离子交换色谱和反相高压液相色谱法从蛋白质水解物中纯化。多肽的氨基酸序列将采用液相色谱-质谱法测定。体外试验将确定将酶（CaMKII、NOS和磷酸二酯酶）活性降低50% （IC50）的抑制剂（肽）浓度。IC50与氨基酸序列之间的关系将使用偏最小二乘回归模型来确定，以提供结构功能特性的新信息，并在食物蛋白质中定位有效的肽序列。动物饲养实验将确定多肽的潜在生理益处。这些实验的结果将确定可用于配制慢性疾病治疗产品的肽，并为加拿大卫生和食品工业提供技术支持。来自结构-功能研究的信息将为科学家提供一个平台，用于未来设计更强大的治疗肽或肽拟物。拟议的工作将导致培养具有食品科学和生命科学跨学科技能的高素质人才。

Previous works from my laboratory have confirmed the feasibility of producing protein hydrolysates that contain low-molecular weight (<1 kDa) calmodulin (CaM)-binding peptides through enzymatic hydrolysis of pea and flaxseed proteins. Due to their small size, the peptides have a high potential to be absorbed intact into the blood circulatory system; in vitro tests confirmed resistance to gastrointestinal digestive enzymes. The protein hydrolysates were shown to inhibit the activities of CaM-dependent metabolic enzymes such as protein kinase II (CaMKII) and nitric oxide synthases (NOS) that have been implicated in the pathogenesis of chronic diseases. The proposed research will use the protein hydrolysates as the raw materials to isolate and identify the structural characteristics of potent CaM-binding peptides. Peptides will be purified from the protein hydrolysates using ion-exchange chromatography and reverse-phase high pressure liquid chromatography methods. Amino acid sequence of peptides will be determined using liquid chromatography-mass spectrometry methods. In vitro tests will determine inhibitor (peptide) concentration that reduces enzyme (CaMKII, NOS, and phosphodiesterase) activity by 50% (IC50). The relationships between IC50 and amino acid sequence of peptides will be determined using partial least square regression modeling to provide new information on structure-function properties and locate potent peptide sequences within food proteins. Animal feeding experiments will determine the potential physiological benefits of the peptides. Results from these experiments will identify peptides that can be used to formulate therapeutic products against chronic diseases and provide technical support for the Canadian health and food industries. Information from structure-function studies will provide scientists with a platform for future design of more powerful therapeutic peptides or peptidomimetics. The proposed work will lead to the training of highly qualified personnel with interdisciplinary skills in Food Science and the Life Sciences.