Bioinformatics Analysis Of Calpain Mediate-cleavage And Phospho-binding

Post-translational modifications orchestrate the biochemical nature of most eukaryote proteins and play significant roles in many biological processes. Although many types of PTMs have been discovered, only a few of them have been well-characterized due to the lack of sufficient data for analysis. However, traditional experimental strategies, such as site-directed mutagenesis, mass spectrometry, peptide library,are labor-intensive and time-consuming. Computational prediction provides an effective approach to conduct preliminary analyses and greatly reduce the number of putative targets which need further experimental verification.In this study, we focus on the bioinformatics analysis of calpain-mediated cleavage and PBD-dependent phospho-binding. Overwhelming research has confirmed that both of them play pivotal roles in numerous biological processes, including the regulation of gene expression, signal transduction and cell cycle progression. clinical evidence suggests that aberrant PTMs are highly associated with diseases. To identify the substrates with bona fide sites and further anatomize the molecular mechanisms of them, we developed GPS-CCD 1.0 for the prediction of calpain cleavage sites and GPS-Polo 1.0 for the prediction of phospho-binding and phosphorylation sites of Plks, respectively. In addition, we annotated potential calpain cleavage sites for hundreds of calpain substrates, for which the exact cleavage sites had not been previously determined. Also, on the basis of currenly available available phosphoproteomics data, we predicted ten thousands of potential phospho-binding and p-sites of Plks in eukaryotes, respectively. Additional sequence analysis together with in vitro and in vivo experimental assays demonstrated that human Mis18B is a novel interacting partner of Plk1, while pT14 and pS48 of Mis18B were identified as phospho-binding sites. Furthermore, statistical analysis suggests that Plks phopho-binding proteins are more implicated in mitosis against its phosphorylation substrates. Taken together, our systematic analysis will propel the research of calpain cleavage and PBD-dependent phospho-binding into a new phase.