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目的研究将热休克蛋白60(HSP60)蛋白序列中第70位的丝氨酸(serine)突变为丙氨酸(alanine)后,HSP60在细胞内定位的改变。方法将PrC/CMV-HSP60P1重组质粒中HSP60P1编码第70位丝氨酸的碱基定点诱变为编码丙氨酸的碱基序列,转染至小胶质细胞BV-2细胞株中表达。细胞经10h缺氧处理后,裂解细胞,应用差速离心方法分离细胞膜、细胞质和线粒体,应用Western-blot方法检测各细胞器中HSP60的含量。结果在转染未诱变PrC/CMV-HSP60P1质粒的细胞中,正常情况下,HSP60大部分分布在线粒体,少部分在胞浆,细胞膜中未见表达;缺氧刺激后,HSP60在细胞膜、胞浆和线粒体中均有表达;然而70位丝氨酸突变为丙氨酸后,只观察到HSP60在胞浆和线粒体中的表达,而在细胞膜上未见表达。结论作为磷酸化位点的Serine70对HSP60在细胞内的定位具有关键作用。
Objective To study the change of intracellular localization of HSP60 after mutating serine from serine to alanine in heat shock protein 60 (HSP60) protein sequence. Methods The base sequence of serine 70 of HSP60P1 in the recombinant plasmid PrC / CMV-HSP60P1 was mutated to a base sequence encoding alanine and then transfected into BV-2 microglia cell line for expression. After 10h hypoxia treatment, cells were lysed and cell membranes, cytoplasm and mitochondria were separated by differential centrifugation. The content of HSP60 in each organelle was detected by Western-blot. Results In the cells transfected with un-mutagenized PrC / CMV-HSP60P1 plasmid, most of the HSP60 were distributed in the mitochondria, but a few were expressed in the cytoplasm but not in the cytoplasm. After hypoxia stimulation, Both plasma and mitochondria were expressed; however, only the expression of HSP60 in cytoplasm and mitochondria was observed after mutation of serine 70 to alanine, but no expression was observed in the plasma membrane. Conclusion Serine70, a phosphorylation site, plays a key role in the intracellular localization of HSP60.