Determination Of Key Amino Acids In P38α - Bypass Activation Inhibitory Peptides And Optimization Of Inhibitory Peptides

Objective:Acute myocardial infarction is usually caused by coronary thrombosis that leads to critical tissue ischemia. Although coronary reperfusion is essential for myocardial salvage, it may at first exacerbate cellular damage sustained during the ischemic period. Myocardial I/R can cause multiple adverse events, such as oxidative stress and intracellular calcium overload, which are responsible for cardiomyocyte death. P38 mitogen activated protein kinase (MAPK) seems to play a causative role in myocardial injury and dysfunction following I/R among the various intracellular signaling pathways activated during this process.As the intervention strategies and drugs to prevent reperfusion injury after an acute myocardial infarction are scant, it is of importance to identify the key sites in this peptide inhibitor of TAB1/p38a interaction. Such efforts might lead to a therapeutic to attenuate such cardiac dysfunction.In this work, on the basis of our previous research, using the distance geometry technology and computer graphics technique according to the 3-D modeling structure of PT5 and p38a, a series of PT5 mutants were designed and evaluated.Methods:1. Theoretical speculation of key sites and synthesize peptide mutantGeometry computer graphics technology was used to identify the key sites in this peptide inhibitor of p38a/TAB1 interaction. Six sequences acquired by substitute every two amino acids for alanines. These six peptides were commissioned to Chinese peptide company to synthesis and purity was more than 95%. In addition, design a control peptide containing 15 amino acid, which does not impact the interaction between p38a/TAB1.2. GST-pull down to identify key amino acidsDissolving GST-TAB1 and His-p38α in Tris buffer and adding alanine substitutions of PT5 mutants to conduct GST pull-down assay. Control peptide as negative control, PT5 as positive control, according to the different ability of impact the interaction between p38α/TABl to determine key amino acid of antagonist peptide.3. In vitro kinase assay to analysis activity of peptidesDissolving GST-TAB1 and His-p38α and ATP in Kinase buffer and adding alanine substitutions of PT5 mutants to conduct in vitro Kinase assay. Control peptide as negative control, PT5 as positive control, according to the different ability of impact the phosphorylation of p38a to determine key amino acid of antagonist peptide.Result and Analysis:A combination of computation techniques and biological mutation have been used to determine the binding site and amino acid residues on the inhibitor peptide that are critical for binding to p38a. Computer-guided ab initio modeling was used to model PT5 and its mutants. Molecular docking simulations using shape descriptions were carried out using the model to determine which residues in the peptide PT5 are involved in binding interaction. Biological mutation of the peptide PT5 was used to evaluate the bio-function when the critical residues were mutated. Base on the biological mutant experimental results, the key residues in PT5, i.e. Thr11 and Asp12, were identified and the core sequence of PT5 was determined. Interestingly, two mutants named PT2 and PT4 showed better effect on both disrupting p38a/TAB1 interaction and inhibiting TAB1 induced p38a phosphorylation. None of the mutants exhibited any effect on MKK6/p38a interaction. The results highlight the key sites and core structure of PT5 binding to p38a, base on which, optimized peptides compounds could be developed for treating myocardial I/R injury in clinical.