Joana Vieira Silva, Ph.D., Maria Joao Freitas, Ph.D., Joana Santiago, M.Sc., Sarah Jones, Ph.D., Sofia Guimaraes, Ph.D., Srinivasan Vijayaraghavan, Ph.D., Steven Publicover, Ph.D., Giorgio Colombo, Ph.D., John Howl, Ph.D., Margarida Fardilha, Ph.D.
To design protein phosphatase 1 (PP1)–disrupting peptides covalently coupled to inert cell-penetrating peptides (CPPs) as sychnologically organized bioportide constructs as a strategy to modulate sperm motility.
Academic research laboratory.
Normozoospermic men providing samples for routine analysis and Holstein Frisian bulls.
Main Outcome Measure(s)
Effect of the bioportides on the activity and interactions of PP1γ2—a PP1 isoform expressed exclusively in testicular germ cells and sperm—and on sperm vitality and motility.
PP1-disrupting peptides were designed based on the sequences from: 1) a sperm-specific PP1 interactor (A kinase anchor protein 4); and 2) a PP1 inhibitor (protein phosphatase inhibitor 2). Those sequences were covalently coupled to inert CPPs as bioportide constructs, which were successfully delivered to the flagellum of sperm cells to induce a marked impact on PP1γ2 activity and sperm motility. Molecular modeling studies further facilitated the identification of an optimized PP1-binding sequence and enabled the development of a modified stop-sperm bioportide with reduced size and increased potency of action. In addition, a bioportide mimetic of the unique 22-amino acid C-terminus of PP1γ2 accumulated within spermatozoa to significantly reduce sperm motility and further define the PP1γ2-specific interactome.
These investigations demonstrate the utility of CPPs to deliver peptide sequences that target unique protein-protein interactions in spermatozoa to achieve a significant impact upon spermatozoa motility, a key prognostic indicator of male fertility.