The Construction Of MDM2 Inhibitors Screening System And Copper Ions Binding Alters The Folding Of N-terminal Of HCTR1

p53 is a tumor suppressor gene,it encodes a protein that plays an important role in the regulation of cell cycle,apoptosis,DNA repair,and angiogenesis.MDM2 can bind to p53 and inhibit its function,it also induces the degradation of p53.Therefore,targeting small molecules to the interaction of MDM2 and p53 to reactivate p53 has become a promising new cancer treatment strategy.These small molecules are called MDM2 inhibitors,they can be identified by various techniques such as surface plasmon resonance(SPR)and fluorescence polarization(FP),the binding constants of MDM2 inhibitors can be measured,too.However,these techniques have some limitations.This paper developed a new small molecular inhibitor screening method-a co-expressed protein complex system,which can quickly identify MDM2 inhibitors,the binding constants of MDM2 inhibitors can be quantified by fluorescence titration.Copper is an essential trace element for almost all living organisms,whereas the level of intracellular copper needs to be tightly regulated.In human cells,copper enters the cell from the cytoplasm by hCTR1.Currently,the three-dimensional structure of hCTR1 solved by electron crystallography indicated the trimeric hCTR1 creates a pore to transport copper.However,the mechanism of which hCTR1 transports Cu(Ⅰ)and the role of its N-terminus is unknown.In this dissertation,HFIP,SDS micelles and DPPC liposomes were used to mimic the cytoplasmic membrane environment,it was found that the secondary structure of hCTR1(1-46)protein in these three membrane environment became better.Under the condition of DPPC,the conformational change after Cu(I)added was comparatively large,which may reflect the natural conformational change of hCTR1(1-46)and Cu(Ⅰ)interaction under physiological conditions.The interaction between hCTR1(1-46)and cell membrane was studied by DPPC phospholipid micelles.Part 1 begins with a literature review of two proteins,including the structure and function of p53 protein,the structure and function of MDM2,the interactions and the structural basis between MDM2 and p53,introduces the research status of inhibitors of MDM2 and p53 protein and the detection methods of MDM2 inhibitors.Then,it introduces the transport pathway and homeostasis of copper,the structure and function of hCTR1 protein,and the recent progress on the interaction between hCTR1 N-terminal and Cu.Part 2 mainly introduces the system construction process of MDM2/p53-GFP protein complexes system.The protein complexes were purified and characterized,and the selectivity and specificity of the protein complex system were verified.The complex system were used to identify the MDM2 inhibitors by naked eyes,MALDI-TOF,SDS-PAGE,and gel filtration chromatography.By excluding the effect of GFP protein on the interaction of MDM2 and p53 protein,and the effect of MDM2 and p53 protein on GFP protein fluorescence,the binding constants of these small molecule and peptide inhibitors were determined by fluorescence titration,they are basically consistent with the binding constants measured by the traditional methods such as FP and SPR in the literature.Therefore,this protein complex system could qualitatively identify the small molecule and peptide inhibitors and could also quantitatively measure the binding constants of the small molecule and peptide inhibitors.In part 3,the interaction between hCTR1(1-46)and Cu(Ⅰ)and Cu(Ⅱ)was studied,the UV titration results showed that the binding constant of hCTR1(1-46)to Cu(Ⅰ)was log K=14.8±0.1.The ESI mass spectrometry revealed that hCTR1(1-46)was capable of coordinating six Cu(I)and three Cu(II).XANES showed that the coordination structure of hCTR1(1-46)and Cu(I)is tetrahedral,and the coordination structure of Cu(II)is a deformed tetragonal structure.According to the literature,SDS micelles,HFIP,and DPPC liposomes were chosen to mimic the plasma membrane environment.These solvent molecules results in the second structure rearrangements of hCTR1(1-46),have less random coil structure,and new a-helical and β-sheet structures appeared.The 1H-15N HSQC spectra of hCTR1(1-46)dispersed better in these solvent.In SDS micelles and HFIP solvent,the conformational change of hCTR1(1-46)reacted with Cu(I)was subtle,but the conformational change under DPPC condition was obvious.It is speculated that this may be more in line with the natural conformational change in the response of the hCTR1 N-terminal protein to Cu(I)under physiological conditions.Meanwhile,DPPC lipsomes was used to study the interaction between hCTR1(1-46)and cell membrane,and it was found that the interaction between hCTR1(1-46)protein and cell membrane became stronger in the presence of Cu(I).