CXC-Mediated Cellular Uptake of Miniproteins: Forsaking “Arginine Magic”

Miniproteins have a size between that of larger biologics and small molecules and presumably possess the advantages of both; they represent an expanding class of promising scaffolds for the design of affinity reagents, enzymes, and therapeutics. Conventional strategies to promote cellular uptake of miniproteins rely on extensive grafting or embedding of arginine residues. However, the requirement of using cationic arginines would cause problems to the modified miniproteins, for example, low solubility in solutions (proneness of aggregation) and potential toxicity, which are open secrets in the peptide and protein communities. In this work, we report that the cell-permeability of cationic miniproteins can be further markedly increased through appending a magic CXC (cysteine-any-cysteine) motif, which takes advantage of thiol–disulfide exchanges on the cell surface. More importantly, we discovered that the high cell permeability of the CXC-appended miniproteins can still be preserved when the embedded arginines are all substituted with lysine residues, indicating that the “arginine magic” essential to almost all cell-permeable peptides and (mini)­proteins is not required for the CXC-mediated cellular uptake. This finding provides a new avenue for designing highly cell-permeable miniproteins without compromise of potential toxicity and stability arising from arginine embedding or grafting