Systematic Identification of Protein Targets of Sub5 Using Saccharomyces cerevisiae Proteome Microarrays.

Pramod Shah,Chien-Sheng Chen

doi:10.3390/ijms22020760

Abstract

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 linear-ized bactenecin variant of bovine. Sub5 is highly effective against fungi with an ability to trans-locate cell membrane, but its targets are unknown. Systematic analysis of Sub5 targets will facil-itate 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 pro-cess of “actin filament-based process”, “actin filament organization”, “actin cortical patch or-ganization”, regulation of “actin filament bundle assembly”. Moreover, the other enriched cat-egories 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 anal-ysis 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 Re-trieval of Interacting Genes/Proteins (STRING) analysis of the protein targets of Sub5 showed ac-tin 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.

Highlights

Introduction nal affiliationsAntimicrobial peptides (AMPs), a hope for the antimicrobial agents, have activities against bacteria (Gram-negative and Gram-positive), fungus, enveloped virus, as well as cancer cells
Streptavidin-DyLight 650 was utilized for the detection of biotinylated Sub5 bound to specific Saccharomyces cerevisiae proteins on Saccharomyces cerevisiae proteome microarrays
We identified, in total, 128 protein targets of Sub5 using Saccharomyces cerevisiae proteome microarrays

Summary

Introduction

Antimicrobial peptides (AMPs), a hope for the antimicrobial agents, have activities against bacteria (Gram-negative and Gram-positive), fungus, enveloped virus, as well as cancer cells. AMPs are small peptide molecules, which are omnipresent in organism tissues, where they play a key role in non-specific defense systems [1]. AMPs are synthesized much earlier than immunoglobulins from adaptive immune response [3]. AMPs are 12 to 46 amino acids long with diverse sequences, structures, and function. The cationic AMPs mostly contain a net positive charge of +2 or more at neutral pH (in presence of arginine or lysine amino acid residues in their sequence) [4] and amphipathic structures with hydrophilic and hydrophobic domain. Cationic AMPs facilitates electrostatic interaction with anionic phospholipids

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Results

Discussion

Conclusion