P53与TRF1、TRF2的体外结合及P53结合功能区的分析

Interaction Between P53 and TRF1、TRF2 and Analysis of Binding Domain of P53 in vitro

通过分析端粒结合蛋白TRF1、TRF2与P5 3的体外结合 ,探讨P5 3 端粒途径调节细胞生命活动的分子机制 .GST和 4种人P5 3 GST融合蛋白经大肠杆菌表达、谷胱甘肽 SepharoseTM4B纯化后 ,进行SDS PAGE和考马斯亮篮染色 .人P5 3包括野生型 (1～ 393)、C端缺失体P5 3N5 (2～ 2 93)、N端缺失体P5 32C(95～ 393)、单个氨基酸突变体P5 3R175H(175R→H) .各纯化蛋白的分子量与预计的完全一致 ,且纯化率达 90 %以上 .将纯化的GST和P5 3 GST融合蛋白与人乳腺癌细胞MCF 7细胞蛋白进行体外结合反应 ,Western印迹检测反应物中P5 3和TRF1、TRF2的结合 .野生型P5 3和P5 3 R175H均能沉淀MCF 7中的TRF1、TRF2 ,且结合力相似 ,而单独的GST则无沉淀TRF1、TRF2的作用 .与野生型P5 3和P5 3R175H相比 ,P5 32C与TRF1、TRF2的结合力明显增加 ,P5 3N5与TRF1、TRF2的结合力大大减弱 .表明P5 3和TRF1、TRF2可以进行直接而特异的体外结合 ,且它们的结合为P5 3C端 (2 93～ 393)结构域依赖性 .P5 3和TRF1、TRF2这种结构域依赖性的结合可能与端粒动态变化所诱导的细胞活动有关 .

To examine the binding of TRFs to series of both unmodified and modified P53 construts in vitro, GST only and 4 different human P53 GST fusion proteins were expressed in E. coli and purified through glutathione Sepharose 4B. P53s were wild type P53 (1～393), N terminal truncated form P53 2C (95～393),C terminal truncated form P53 N5 (2～293) and single amino acid mutant P53 R175H (175 arginine to histidine). SDS PAGE and Coomassie brilliant blue staining showed that the molecular weights of all the purified proteins were as expected and purities were over 90%. Purified GST and P53 fusions were mixed with human breast cancer cell MCF 7 cellular protein extracts through in vitro binding assay pull down, the molecular interaction between P53 and TRF1,TRF2 were detected by Western blotting, respectively.The results showed that both wild type P53 and P53 R175H could bind with TRF1,TRF2 of MCF 7 cells, their binding capacities were similar, whereas GST alone did not. Comparison of the above individual binding capacity with wild type P53 revealed that the interaction between P53 2C and TRF1,TRF2 enhanced dramatically, Whereas between P53 N5 and TRF1,TRF2 reduced significantly, suggesting that P53 could interact with TRF1,TRF2 directly and specifically in vitro, C terminus of P53 (293～393) was the binding domain of their interaction. This C terminus domain dependent interaction between P53 and TRF1,TRF2 may be related to the cellular activities induced by telomere dynamic changing.