Hsp27 Interacts With Protein-L-isoaspartyl Methyltransferase (PIMT) In Breast Cancer Cells

Breast cancer is one of the most common cancer of women. Its recurrence and metastasis is the leading cause of death in breast cancer patients. As a small molecular chaperone, Hsp27 plays an important role in invasion,metastasis and resistance of breast cancer, but the regulatory mechanism is unclear. To study the molecular mechanism of Hsp27 in breast cancer cells, Our laboratory have identified 17 Hsp27-associated proteins in MCF-7L cells using Hsp27 immunoprecipitation assay coupled with Two-Dimensional Gel Electrophoresis and Mass Spectrometry analysis, One of them is Protein L-isoaspartyl Methyltransferase (PIMT). PIMT is a protein repair enzyme, also involved in cell stress response as with the Hsp27. Studies have shown that Hsp27 and PIMT are both involved in regulation of PI3K/Akt and MAPK signaling pathway and the regulation of epilepsy. These suggest they may play an important function in the mechanism of the disease. Hence, further study of the relationship between PIMT and Hsp27 is significant to clarify the molecular mechanism of breast cancer and search for new Diagnostic markers and therapeutic targets.This study aims to verify the interaction of Hsp27 with PIMT and explore their regulatory mechanism in breast cancer cells.The recombinant expression vector pGEX-4T-1-Hsp27 with the GST tag was constructed in the method of gene-recombination. The recombinant expression vector GST-Hsp27 and 6×His-PIMT were expressed,purified and then identified by SDS PAGE and Mass Spectrum( MS ). The in vitro interaction between GST-Hsp27 and 6×His-PIMT was examined by means of GST pull-down assay as well as Western Blot. In order to examine their colocalization in the cell line of MCF-7L through the technique of Confocal Laser Scanning Microscope. The specific primary antibody and second antibody with a fluorensence label Alexa Fluor 546/488 were used in the immunization double fluorenscence stainning of endogenous Hsp27 and PIMT . The recombinant expression vector pGEX-4T-1-Hsp27 was successfully constructed. GST-Hsp27 or 6×His-PIMT were purified and identified by SDS-PAGE as well as MS, and their direct interaction in vivo were evidenced by GST pull-down. The co-localization of Hsp27 and PIMT in the cytoplasm of the cell line MCF-7L was observed by Confocal Laser Scanning Microscope.Our studies indicated that PIMT is a Hsp27 binding protein and suggest their interaction may play an important role in breast cancer cells.