Antimicrobial peptides (AMPs) are intensively studied in terms of alternative drugs. Sub5 is a synthetic 12-mer AMP with substitutions of five amino acids of bactenecin 2A (Bac2A), a linearized bactenecin variant of bovine. Sub5 is highly effective against fungi with an ability to translocate cell membrane, but its targets are unknown. Systematic analysis of Sub5 targets will facilitate our understanding on its mechanism of action. In this study, we used high-throughput Saccharomyces cerevisiae proteome microarrays to explore the potential protein targets of Sub5. The screening results showed 128 potential protein targets of Sub5. Bioinformatics analysis of protein targets of Sub5 revealed significant gene ontology (GO) enrichment in actin related process of “actin filament-based process”, “actin filament organization”, “actin cortical patch organization”, regulation of “actin filament bundle assembly”. Moreover, the other enriched categories in GO enrichment mostly contained actin associate proteins. In total, 11 actin-associated proteins were identified in the protein targets of Sub5. Protein family (PFAM) enrichment analysis shows protein domain enriched in actin binding, i.e., “Cytoskeletal-regulatory complex EF hand (helix E-loop-helix F motif)”. Being consistent with GO analysis, Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis of the protein targets of Sub5 showed actin network with involvement of 15 protein targets. Along with actin-network, STRING analysis showed protein–protein interaction network in ribonucleoprotein, transcription and translation, chromosome, histone, and ubiquitin related, DNA repair, and chaperone. Multiple Expression motifs for Motif Elicitation (MEME) suite provided a consensus binding motif of [ED][ED]EEE[ED][ED][ED][ED][ED], in total of 75 protein targets of Sub5. This motif was present in 9 out of 15 actin-related proteins identified among protein targets of Sub5.
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