Disruption of protein phosphatase 1 complexes with the use of bioportides as a novel approach to target sperm motility

Objective: 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. Design: Experimental study. Setting: Academic research laboratory. Patient(s)/Animal(s): Normozoospermic men providing samples for routine analysis and Holstein Frisian bulls. Intervention(s): None. 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. Result(s): 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. Conclusion(s): 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.