The Study Of The Polypeptide Ligand Of RIP3 Protein And The Quantitative Method Of Polypeptide Synthesis Yield

Objective: In multiple pathways of cell necroptosis,DAI protein,TRIF protein and RIP1 protein can interact with RIP3 proteins to form a similar death complex,indicating that RIP3 protein is the key protein of programmed necrosis,while RIP3 and other proteins are through Rhim Domain domains interact,and it can be seen that Rhim domain is necessary for interaction between RIP3 and RIP1 proteins.Previous studies have identified two small molecules that inhibit cell necrosis,respectively Nec-1 and NSA,but their target sites are MLKL proteins in the lower reaches of RIP1 and RIP3,which do not directly affect the key protein RIP3.Therefore,we choose the RIP3 protein as the target,and find the polypeptide ligand in vitro in order to inhibit the programmed necrosis of the cells so as to cure the necrosis-related diseases.Methods: By using solid phase synthesis,reversed phase high performance liquid chromatography purification,Matrix assisted laser dissociation Time-of-flight mass spectrometry to identify a series of peptides containing rip1-rhim sequences,the fluorescence polarization analysis method was used to detect the activity of the binding RIP3 protein,and the Kd value was calculated by fitting curve.The sequence of the binding activity can be screened by truncating the N-and C-terminal of the four key amino acid residues without changing them.It is possible to form the Q-amino acid in the mutant polypeptide ligand,and to compare the effect of peptide ligand binding activity and to improve the stability of polypeptide ligand.After the modification of polypeptide ligand skeleton,HP series polypeptide was compared,and whether the peptide ligand binding activity was affected and the stability of polypeptide ligand could be improved.Results: 1)To verify the feasibility of the method,selection of 9 natural sources of polypeptide ligand,according to the fluorescence polarization method analysis of the binding activity of different peptides,found that different peptides and RIP3 protein binding ability is not the same,hydrophobic groups of the same size polypeptide in the combination with RIP3 protein performance better.2)The AC-HP2 competition FITC-HP2 experiment verifies that the binding of polypeptide and protein is reversible.3)from N-terminal to h RIP3 polypeptide ligand truncated,greatly affected its binding activity with RIP3 protein,from the C-terminal truncated to its RIP3 protein binding activity has a certain effect.4)replaces Q with a without charge,there was no significant effect on the binding activity of h RIP3,while the substitution Q was positively charged or negatively charged K or D,which affected the original binding activity of h RIP3,and the substitution Q for A,D,K did not significantly help the stability of polypeptide.5)After the polypeptide skeleton was modified to get HP series polypeptide,the binding activity of RIP3 protein decreased slightly.HP Series Polypeptides are combined with the RIP3 protein in accordance with 1:1.HP series polypeptide ligands are much more stable than h RIP3 peptides,of which HP2,HP2' perform well.