Strategies For Restoring Activity Of P53 From Nonsense Mutation And Inhibiting Inactivation

p53 protein is an important tumor suppressor and plays an important role in cell cycle arrest,DNA damage repair,and apoptosis.Nearly half of malignant tumors are associated with p53 mutations,and another half of tumors are associated with the inhibition of p53 activity.Recovery of p53 activity has been proven to be an effective treatment for tumors or preventing their metastasis.Therefore,it is of clinical significance to restore the activity of endogenously inactivated p53.Premature stop codons(PTCs)will lead to the generation of truncated p53 protein,which such the nonsense mutation p53 is highly associated with cancer.It has been demonstrated that aminoglycoside molecules are able to induce PTC readthrough and restore functional full-length p53.However,there is currently a shortage of high-throughput screening methods for small molecules that enable rapid and efficient reading through of p53.In this study,we set up an effective screening method for screening small molecule to read through the nonsense mutation p53.This method utilized the fusion gene of p53-PTC and green fluorescent protein(GFP)which expressed in E.coli cells and H1299 cells.Our data revealed that after treatment of cells with aminoglycosides,full-length p53 protein could enable fluorescence detection of the GFP protein.This model can be extended to screen aminoglycoside antibiotics and their analogues,small molecules to read through premature stop codons.Furthermore,we found that cancer cells after treated with low-dose doxorubicin in combination of and geneticin(G418)can achieve the same effects as using high concentration of doxorubicin alone.The discovery has provided a new therapeutic strategy for cancer treatment.On the other hand,we explored whether that p28 and N24 polypeptides have the effect of suppressing p53 activity.Previous studies have found that p28 can target cancer cells with anti-proliferative activity and pro-apoptotic activity.p28 interacts with p53 DBD(DNA binding domain)to reduce p53 degradation and enhance p53 activity.However,the mechanism of its combination is not yet clear.N24 is a polypeptide that promotes cell cycle arrest and has anti-tumor activity.We found that N24 interacts with p53 DBD.In this study,we would like to structural biology to analyze the mechanism of interaction between p28/N24 and p53 DBD.We have screened the crystallization conditions of the p28 and p53 DBD complexes and the N24 and p53 DBD complexes and have obtained the crystals of the complexes.