Disruption of Protein Phosphatase 1 complexes using bioportides as a novel approach to target sperm motility

Abstract

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. Patients/Animals: 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. Results: PP1‐disrupting peptides were designed based on the sequences from (i) a sperm-specific PP1 interactor (A kinase anchor protein 4, AKAP4) and (ii) a PP1 inhibitor (protein phosphatase inhibitor 2, PPP1R2). 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 upon PP1γ2 activity and sperm motility. Molecular modelling studies further facilitated the identification of an optimized PP1 binding sequence and enabled the development of a Modified Stop Sperm (MSS1) bioportide with reduced size and increased potency of action. Additionally, 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: These investigations demonstrate the utility of CPPs to deliver peptide sequences 53 that target unique protein-protein interactions in spermatozoa to achieve a significant impact upon 54 spermatozoa motility, a key prognostic indicator of male fertility.