De-ubiquitination The Metal To Protease Brcc36 And Related Protein In Breast Cancer Dna Damage Repair Pathway Gene Cloning, Expression, Purification And Characterization Of

Breast cancer is one of the common malignant tumors of women, which seriously threaten women's health. About 1,000,000 women suffer from breast cancer every year all over the world. In China, however, women run a lower risk of it, the rate of breast cancer is been significantly increasing in recent years, ranking second in the cancer of women. The causes of it are complex, which containing genetic factors, environment and habits, among which genetic factors play a major role in the development and progression of breast cancer. Different breast cancers have different treatments. Currently, BRCA1, BRCA2, P53, CHEK3, ATM, NBSI, BRIPI and PALB2 have been confirmed to be breast cancer susceptibility genes, and of which BRCA1 and BRCA2 are the most important ones. About 40% hereditary breast cancers relate to mutation of BRCA1 and BRCA2. Moreover, these proteins are involved in many cellular processes, including DNA repair and cell cycle checkpoint control. BRCA1 and BRCA2 together with other proteins form a larger complex called BRCC, which regulate DNA repair. BRCC36, a deubiquitinating metalloenzyme of the BRCC complex, directly interacts with BRCA1, which aberrant expressed in most breast cancers. Knock-down of brcc36 alone does not affect cell growth, but when combined with ionizing radiation (IR) exposure, it leads to an increase in the percentage of cells undergoing apoptosis. BRCC complex is an ubiquitin E3 ligase, which connects a group of ubiquitin to P53, and then ubiquitination of P53 will be degraded in proteasome. The mechanism of ubiquitination and deubiquitination regulated by BRCC complex is still not clear. Here, we get sufficient soluble BRCC36 protein, BRCAl502-1054 protein, P53 protein from Escherichia coli. We studied the role of Zn2+ in maintaining the secondary structure of BRCC36 by Circular dichroism (CD) and the interaction between Zn2+ and BRCC36, Zn2+ and P53, and BRCC36 and P53 by isothermal titration calorimetry (ITC).The results show Zn2+ plays a key role in maintaining the secondary structure of BRCC36, Zn2+ interacts with both BRCC36 and P53, which combines at the ration of 1:1. P53 and BRCC36 have no thermal effect, indicating BRCC36 requires other subunits involved directly to make P53 deubiquitination to maintain the stability of P53. This mechanism remains to be further studied.