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

钙调素（CaM）是一种分子量为16.7 kDa的多功能钙结合蛋白，是细胞内钙信息的主要翻译者。钙调素是钙依赖性控制许多细胞活动，包括细胞增殖，细胞分裂和神经传递所必需的。钙调素依赖性酶的过度活性可导致代谢紊乱，如糖尿病、高血压、细胞生长异常和关节炎。因此，与CaM结合并抑制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作或进展。我以前的NSERC资助的工作导致了几种食物（亚麻籽和黄豌豆）蛋白衍生的肽序列的发现，这些肽序列作为钙调素依赖性酶（如磷酸二酯酶1（CaMPDE）和一氧化氮合酶（NOS））的体外抑制剂。因此，本发明的主要目的是确定这些肽（2-7个氨基酸残基）逃避胃肠道消化并被完整吸收到血液循环系统中的能力。这项工作还将确定吸收的肽对CaMPDE和NOS酶（内皮，神经元和诱导型）的组织活性的抑制能力，包括肽诱导的内源性毒性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，将向Wistar大鼠喂食具有已知肽组成的纯化肽或蛋白水解产物级分。灌胃后，使用液相色谱/质谱技术测定血液的肽组成，以确定是否发生吸收。口服肽的大鼠进行乙醇或脂多糖诱导的氧化应激将随后测定血浆和器官组织水平的有毒自由基，特别是超氧化物的生产（由内皮NOS催化）变得大大增强在氧化应激。拟议的工作可以促进新型肽基补充剂的开发，这些补充剂可用作预防体内有毒自由基积累的预防剂。

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.