EF-hand对钙调磷酸酶B同源蛋白2的生物学功能影响

摘要

目的 探讨EF-hand结构对钙调磷酸酶B同源蛋白2(CHP2)的生物学功能的影响.方法 通过PCR扩增CHP2 cDNA片段,构建带GFP标签的野生型及EF-hand突变型CHP2表达质粒;与钠氢离子交换蛋白1(NHE1)共转染PS120细胞;采用激光共聚焦显微镜、膜滤过法、免疫共沉淀等手段,明确CHP2结合Ca2+的数量、部位,以及对CHP2与NHE1结合的影响;在血清饥饿条件下,观察表达EF-hand突变的CHP2(NHE1/CHP2-EF3m/4m)对细胞生存的影响.结果 分别突变EF-hand结构EF3或EF4,CHP2结合Ca2+减少;同时突变EF3和EF4,CHP2不能结合Ca2+;免疫共沉淀实验结果显示乙二胺四乙酸(EDTA)剥夺CHP2携带的Ca2+之后,CHP2与NHE1结合的数量明显减少;不携带Ca2+的CHP2在细胞内与NHE1结合的数量也明显减少;在血清饥饿条件下,表达不携带Ca2+的CHP2的细胞存活数量减少.结论 CHP2能结合2个Ca2+,结合部位在EF3和EF4;携带Ca2+能增强CHP2与NHE1的结合能力;在血清饥饿条件下,携带Ca2+的CHP2能提升细胞抵抗死亡的能力.%Objective To investigate the roles of EF-hand motifs in the regulation of the function of CHP2. Methods The CHP2 EF-hand motifs variants were amplified by PCR based strategy. Recombinant plasmids of GFP-tagged CHP2 and its EF variants were constructed and transfected into PS120 cell and stable clones were selected with G418. Fluorescence microscope was used to detect the subcellular distribution of CHP2 and its variants. The binding capacity of Ca2+ to various CHP2 mutant proteins was measured by a membrane filtration procedure. Co-immunoprecipitation was performed to detect that whether the binding of CHP2 and NHE1 was affected by Ca2+. The survival state of the cells co-expressing NHE1/ CHP2 or NHEl/CHP2-EF3m/4m under serum starvation was observed. Results While the EF3 and EF4 were mutated respectively, the Ca2+ binding to CHP2 was found decreased and Ca2+ could not bind to CHP2 when the two EF-hand motifs were mutated at the same time. CHP2 was found could not interact well with NHE1 with Ca2+ chelated by EDTA according to the results of Co-immunoprecipitation. The binding capacity of EF-hand mutated CHP2 with NHE1 was found decreased in the cells. Lower viability was found in serum starvation according to cells co-expressing NHE1/CHP2-EF3m/ 4m. Conclusion The CHP2 can bind two Ca2+ with two EF-hand motifs (EF3 and EF4); Ca2+ enhances the combination of NHE1 and CHP2. Binding of two calcium ions to the corresponding EF-hand motifs of CHP2 protein increases cell resistance to the death induced by serum starvation.