based on proteomic analysis, including a fourth-generation Group-based Prediction System (GPS) algorithm (Ren et al., 2008). The latest training data set contains 583 palmitoylation sites from 277 proteins, experimentally proved and reported in scientific literature. Particle Swarm Optimize (PSO) was also integrated to improve the convergence speed and training accuracy. The prediction performance and system robustness of CSS-Palm 4.0 was evaluated by the leave-one-out validation and 4-, 6-, 8-, 10-fold cross-validations. To further uncover the mechanism underlying the association between INCENP and ZDHHC5 expression, we analyzed the S-palmitoylation sites of INCENP using CSS-Palm 4.0. As shown in Figure 4A, INCENP has 7 cysteine sites predicted to be S-palmitoylated. Among them, Cys15 had the highest prediction score of 23, and the scores of other sites were less than 8. Actually, the first 26 amino acids are a non-conserved sequence. This peculiarity makes it difficult to predict its effect on protein structure. In this regard, we analyzed the Cys15 site, and found that it was a conserved amino acid site among species, as illustrated in Figure 4B. Taken together, the results indicated that Cys15 may be the key site for palmitoylation of INCENP, as well as for targeting centromeres.
