Discrete arginine topologies guide escape of miniature proteins from early endosomes to the cytoplasm
Polypeptides and peptide mimetics sample a wide chemical space with broad potential to modulate cellular function, but their application to cytoplasmic targets is limited because when added to cells their cytosolic concentration remains low. This limitation is due to a diffusion barrier (the plasma membrane) and absence of dedicated import machinery. Highly cationic peptides and proteins sometimes gain cytosolic access, but how they do so is not well understood. Using a small library of cationic miniature proteins, I probe the influence of positive charge number and orientation on the ability the miniature protein to access the cytoplasm. Using a novel assay, I identify a cationic miniature protein, which we called 5·3, that carries a discrete arginine motif and efficiently reaches the cytoplasm. Database searches find that the precise motif identified (an arginine present in positions i, i + 4, i + 7, i + 10, and i + 11 of an alpha-helix) is not present in nature, but that similar motifs are present in natural proteins that interact with cellular membranes. Finally, I examine the cellular pathway by which 5·3 reaches the cytoplasm. I find that this miniature protein enters the cell via a dynamin and cholesterol dependent endocytic mechanism and is delivered to Rab5+ early endosomes. In contrast to the shiga-like toxins, and many non-enveloped viruses (which escape to the cytoplasm from the endoplasmic reticulum) as well as other peptides previously identified as 'cell penetrating', only 5·3 escapes from early endosomes. These findings should enable the future dissection of the precise molecular events underlying cytoplasmic access of peptides and proteins, and may illuminate principles for the engineering of peptides and peptidomimetics that access cytoplasmic targets.