Structure-function properties of food protein-derived calmodulin-binding peptides: in vivo studies

钙调蛋白（Calmodulin, CaM）是一种16.7 kDa的多功能钙结合蛋白，是细胞内钙信息的主要翻译蛋白。CaM对许多细胞事件的钙依赖性控制是必需的，包括细胞增殖、细胞分裂和神经传递。cam依赖性酶的过度活动可导致代谢紊乱，如糖尿病、高血压、异常细胞生长和关节炎。因此，结合CaM并抑制CaM依赖性酶活性的肽可用于配制营养补充剂，以预防慢性疾病的发生或进展。我之前的nserc资助的工作导致发现了几种食物（亚麻籽和黄田豌豆）蛋白质衍生的肽序列，这些序列在体外作为cam依赖性酶的抑制剂，如磷酸二酯酶1 （CaMPDE）和一氧化氮合酶（nos）。因此，本研究的主要目的是确定这些肽（2-7个氨基酸残基）在胃肠道中逃避消化并被完整地吸收到血液循环系统的能力。这项工作还将确定吸收肽对CaMPDE和NOS酶（内皮酶、神经元酶和诱导酶）组织活性的抑制能力，包括肽诱导的内源性有毒自由基水平的降低以及抗氧化能力水平的提高。为了确定体内吸收特性，Wistar大鼠将被喂食纯化肽或含有已知肽成分的蛋白质水解物。灌胃后使用液相色谱/质谱技术测定血液中的肽组成，以确定是否发生了吸收。对乙醇或脂多糖诱导的氧化应激大鼠口服多肽，随后测定血浆和器官组织中有毒自由基的水平，特别是在氧化应激期间产生的超氧化物（由内皮细胞NOS催化）大大增加。这项工作可以促进新型肽类补充剂的开发，这些补充剂可以用作预防体内有毒自由基积累的药物。

Calmodulin (CaM) is a 16.7 kDa multifunctional calcium-binding protein that is a proven major translator of intracellular calcium message. CaM is necessary for calcium-dependent control of many cellular events including cell proliferation, cell division and neurotransmission. Excessive activities of CaM-dependent enzymes can lead to metabolic disorders such as diabetes, hypertension, abnormal cell growth and arthritis. Therefore, peptides that bind to CaM and inhibit activity of CaM-dependent enzymes may be used to formulate nutritional supplements for preventing onset or progression of chronic ailments. My previous NSERC-funded work led to the discovery of several food (flaxseed and yellow field pea) protein-derived peptide sequences that work as in vitro inhibitors of CaM-dependent enzymes such as phosphodiesterase 1 (CaMPDE) and the nitric oxide synthases (NOSs). Therefore, the main objective of current proposal is to determine ability of these peptides (2-7 amino acid residues) to escape digestion in the gastrointestinal tract and be absorbed intact into the blood circulatory system. The work will also determine inhibitory ability of the absorbed peptides against tissue activities of CaMPDE and NOS enzymes (endothelial, neuronal and inducible), including peptide-induced reductions in levels of endogenous toxic free radicals as well as increased level of antioxidant capacity. To determine in vivo absorption properties, Wistar rats will be fed purified peptides or protein hydrolysate fractions with known peptide compositions. Peptide composition of the blood will be determined after gavage using liquid chromatography/mass spectrometry techniques to determine if absorption occurred. Oral feeding of peptides to rats subjected to ethanol- or lipopolysaccharide-induced oxidative stress will be followed by determination of plasma and organ tissue levels of toxic free radicals, especially superoxide whose production (catalyzed by endothelial NOS) becomes greatly enhanced during oxidative stress. The proposed work could enhance development of novel peptide-based supplements that can be used as preventive agents against accumulation of toxic free radicals in the body.