中华鳖肌酸激酶（PSCK）对应激反应的响应机制研究

The Chinese softshell turtle (Pelodiscus sinensis) is one of the most famous nourishing treasures in China, but it is very sensitive to stress during cultivation. The body's energy metabolism disordered during the stress process, and the creatine kinase (CK) was crucial to maintain the stability of the intracellular ATP. However, the regulation mechanism of CK is not reported in turtles to date...Recently, we found that three kinds of isoforms of CK in Chinese soft-shelled turtle and they were differentiated from CKs of mammals or birds. In order to study the regulatory mechanism of P. sinensis creatine kinase (PSCK) in ATP metabolism during the stress process, the following studies might be carried out: the followings are to be carried out: (1) revealing the differences of PSCK isoforms with respect of structure and function by studying computational modeling and enzyme kinetics; (2) probing the specific binding proteins of PSCKs by conducting yeast two-hybridization and protein-protein interaction predictions; (3) exploring histologic changes of muscle fibers and expression and distribution of PSCKs at the levels of microstructure and ultrastructure; (4) detecting protein and mRNA expressions changes of PSCKs during the stress-derived cultivations; (5) Studying the expression pattern of PSCKs-binding proteins and finally, summarized the regulatory mechanisms in the response of stress . This project would be an important basic researche for understanding the energy metabolism changes of Chinese soft-shelled turtle during the stress conditions.

中华鳖是我国独具特色的水产养殖品种，但是对应激反应敏感。在应激发生时机体能量代谢失调，肌酸激酶（CK）对维持细胞内ATP的稳定起到了关键作用，然而龟鳖类动物中CK的应激响应机理还未见报道。..前期研究中我们首次发现中华鳖肌肉中同时存在三种PSCK 亚型。为了研究中华鳖CK（PSCK）在应激过程中的响应机理，本项目拟开展如下研究：（1）通过结构建模和酶动力学研究来揭示PSCK亚型结构和功能上的差异；（2）通过计算机蛋白相互作用预测以及酵母双杂交实验，筛选出PSCK特异结合蛋白；（3）从显微和超微水平来研究应激前后肌纤维组织学变化、PSCK蛋白定位变化，以及应激前后核酸与蛋白表达量的变化；（4）对PSCK特异结合蛋白的表达规律进行研究，以期找到三种PSCK亚型对应激的响应机理。该项目对于探索龟鳖类动物的能量代谢也具有重要的理论价值。

中华鳖是具有冬眠特性的爬行动物，对应激反应敏感。本项目以中华鳖肌酸激酶（PSCK）为研究对象，研究了PSCK对应激反应的响应。主要结果如下：（1）对三种PSCK的克隆和表达，结果发现同时在心肌和骨骼肌中表达，这是中华鳖特有的表达方式，可能对其特殊的能量代谢具有生物学意义；（2）对三种PSCK的高级结构进行模拟，发现ATP结合区的空间结构非常相似；（3）中华鳖心脏成纤维细胞在受到氧化应激时，PSCKB是应激响应的主要亚型；（4）过氧化氢（H2O2）体外氧化应激PSCK时，直接作用于PSCKM半胱氨酸活性位点，阻碍ADP和活性中心的结合，从而使酶活性丧失；（5）重金属Cd2+和Cu2+结合PSCK后，阻碍ADP和活性中心的结合，从而使酶活性丧失，甘氨酸可脯氨酸可以使失活的酶部分恢复活性；（6）通过酵母双杂交筛选PSCK结合蛋白，GAPDH、NDUFS3，以及ALDOA（4.1.2.13）蛋白，这几个蛋白可能在调节PSCK活性中发挥了关键作用；（7）通过建立中华鳖免疫应激模型，PSCKB可能发挥了重要的能量调节作用，在脾脏中表达量迅速上升；（8）通过建立冬眠低温应激模型，结果发现在肌肉和心脏中显著降低的亚型是PSCKS，而脑组织和血液中显著降低的亚型是PSCKB和PSCKM。PSCK沟通了呼吸链（线粒体）——糖酵解（细胞质）——ATP消耗（细胞质）之间磷酸基团的转移，对于中华鳖应激反应时的能量代谢具有关键调节作用。