BIOTECHNOLOGICAL POTENTIAL OF ANIONIC AND AMPHIPHILIC ALPHA-HELICES ENCRYPTED IN THE HUMAN PROTEOME

Membrane-active peptides can be arranged in three distinct classes: cell-penetrating peptides, amyloids, and antimicrobial peptides. Each class represents a promising alternative for treatment of diseases by means of drug delivery, prevention, or killing of microorganisms. Membrane-active peptides are mostly cationic and amphiphilic. However, research on anionic membrane-active peptides is gaining traction in the scientific community, particularly with peptides from the GALA and pHLIP families, which interact with membranes in a pH-sensitive manner. Following methodologies employed in previous works in our laboratory, anionic intragenic peptides capable of adopting amphiphilic helical structures were screened in the human proteome. Peptide HsIDP1 was synthesized as proof of concept to assess its activity against biological membranes. Circular dichroism assays revealed the peptide's pH-sensitive activity in acidic environments, akin to pHLIP, due to the presence of acidic side chains that, when deprotonated, hinder α-helix formation and membrane insertion. Studies using large unilamellar vesicles (LUVs) composed of DMPC/DMPG 2:1 (w/w) showed that the vesicles' anionic nature promoted peptide structuring in contrast to zwitterionic vesicles. Finally, studies on cell viability and antimicrobial activity demonstrated that HsIDP1 exhibited cytotoxicity towards BV-2 cells only at high concentrations, while it failed to inhibit microbial growth at physiological pH. The present work represents the first steps in the systematic prospection of anionic and amphiphilic segments of human proteins and their biotechnological use as pH-sensitive membrane translocation agents.