[The molecular interaction between P53 and telomeric repeat binding protein 1 in vitro].
OBJECTIVE
Cellular proliferate inhibition, senescence or apoptosis are induced by telomere shortening through the activation of P53 pathway, but so far, little is known of the mechanism. This study aimed to clarify the molecular regulation of P53 through telomere pathway by the investigation of molecular interaction between P53 and the main telomere associated protein telomeric repeat binding protein 1(TRBP1) in vitro.


METHODS
Glutathione S-transferase (GST) alone and 4 different human P53-GST fusion proteins were expressed in E.coli. and purified through glutathione Sepharose (TM)4B by affinity chromatography, 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). Glutathione Sepharose (TM)4B, purified GST alone and P53 fusions were mixed with human breast cancer cell line MCF-7 cellular protein extracts through in vitro binding assay-pull down, the molecular interaction between P53 and TRBP1 were detected by Western blot.


RESULTS
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%. Western blot of TRBP1 showed that both wild type P53 and P53 R175H could bind to TRBP1 of MCF-7 cells, and their binding capacities are similar, whereas GST alone and Glutathione Sepharose(TM) 4B beads could not. Compared with both of them, the interaction between P53 2C and TRBP1 enhanced dramatically, but between P53 N5 and TRBP1 reduced significantly.


CONCLUSIONS
P53 can interact with TRBP1 directly and in vitro, C terminus of P53 (293-393) is the structural domain of their interaction. This C terminus domain dependent interaction between P53 and TRBP1 may be related to the cellular activities induced by telomere dynamic changing.
Department of Pathology, Peking University School of Basic Medical Sciences, Beijing 100083, China. lingli@bjmu.edu.cn
Ling
L
Li
Bo
B
Zhang
Wan-zhong
WZ
Zou
Jie
J
Zheng
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D017209:Apoptosis; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D035321:Telomeric Repeat Binding Protein 1 / Q000235:genetics / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D035321:Telomeric Repeat Binding Protein 1; D016159:Tumor Suppressor Protein p53; D005982:Glutathione Transferase