糖酵解酶N端丝氨酸基团磷酸化调控钙蛋白酶活性机理

Tenderness is the most important eating quality of meat, Calpain has been considered a key endogenous tenderizing enzymes, and was influenced by pH or calcium ion which were indirectly regulated by glycolytic enzymes. On the other hand, some studies had shown that serine phosphorylation of glycolytic enzymes are directly involved in the activity of calpain, which effects on tenderness of meat, but the specific mechanism is unclear. The project intends to systematically study the effect of phosphorylation on the functional and structural characteristics of glycolytic enzymes during the aging of post-mortem muscle, identify the typical glycolytic enzymes, and confirm serine phosphorylation sites, the protein conformation and the activities to glycogen metabolites. Furthermore, to investigate the bingding between glycolytic enzymes and sarcoplasmic reticulum calcium pump protein, the degradation of calpain to glycolytic enzymes which are phosphorylated,and to study the effect of serine phosphorylation glycolytic enzymes on the structure and function of calpain , calpastatin and calpain activator. Based on assays, to reveal the regulatory mechanism of calpain by serine phosphorylation of glycolytic enzyme directly during the post-mortem aging. The mechanism will be investigated by phosphoproteomics, Western blotting, Yeast Two-hybrid,immunoprecipitation, immunohistochemistry phosphorylation, transform infrared spectroscopy and circular dichroism techniques. Finally, the research results will improve and develop the theory for muscle tenderness.

嫩化决定肉品品质，经典的钙蛋白酶嫩化理论认为，糖酵解产生乳酸降低肌肉pH值、促进钙离子释放，激活钙蛋白酶活性进而间接调控肌肉嫩度，而近期研究发现糖酵解酶磷酸化也直接参与钙蛋白酶活性的调控，影响肌肉嫩化，但具体机理尚不清楚。本项目以嫩度差异显著的牛羊肉为试验材料，采用磷酸化蛋白质组学、磷酸化蛋白质免疫印迹、酵母双杂交、磷酸化免疫共沉淀等技术，分离、鉴定差异显著的磷酸化糖酵解酶，确证其丝氨酸磷酸化位点、活性和结构变化；考察糖酵解酶磷酸化后与肌浆网、肌动蛋白细丝的结合情况，以及两者结合后对钙离子释放的影响；研究钙蛋白酶对磷酸化糖酵解酶的竞争性降解作用，糖酵解酶磷酸化对钙蛋白酶、钙蛋白酶抑制蛋白、钙蛋白酶激活蛋白的作用。确证影响钙蛋白酶活性的磷酸化糖酵解酶种类、磷酸化位点及其结构与功能变化，揭示糖酵解酶N端丝氨酸基团磷酸化调控钙蛋白酶活性的机理，丰富和发展肌肉钙蛋白酶嫩化理论。

嫩化决定肉品品质，经典的钙蛋白酶嫩化理论认为，糖酵解产生乳酸降低肌肉pH值、促进钙离子释放，激活钙蛋白酶活性进而间接调控肌肉嫩度，而近期研究发现糖酵解酶磷酸化也直接参与钙蛋白酶活性的调控，影响肌肉嫩化，但具体机理尚不清楚。本项目研究了不同嫩度羊肉中磷酸化差异的糖酵解酶及其磷酸化方式，糖酵解酶磷酸化水平与μ-钙蛋白酶活性及钙蛋白酶抑制蛋白降解之间的关系，确证了糖酵解酶磷酸化对μ-钙蛋白酶活性的影响，揭示了不同温度、钙离子浓度条件下糖酵解酶磷酸化对μ-钙蛋白酶活性和二级结构的直接调控机制以及磷酸化介导的钙蛋白酶抑制蛋白对μ-钙蛋白酶活性的间接调控机制。结果表明，糖酵解酶磷酸化水平与羊肉嫩度负相关，糖酵解酶磷酸化水平升高通过抑制μ-钙蛋白酶自溶和降解过程对μ-钙蛋白酶的活性起负向调控作用，冰温（-1℃）和4℃条件下低磷酸化组μ-钙蛋白酶的降解率最高，钙离子浓度过高时会削弱磷酸化对μ-钙蛋白酶活性的影响，揭示了蛋白激酶A催化μ-钙蛋白酶磷酸化后可使μ-钙蛋白酶更易被钙蛋白酶抑制蛋白抑制的调控机制。上述研究成果为研发肉品嫩度调控技术提供了理论支撑。