Rapid Uptake and Ubiquitination of Fluorescent Peptides into Mammalian Cells Using a β-Hairpin Sequence Motif

Fluorescent peptide-based reporters have emerged as a prominent method to quantify intracellular enzyme activity in cells. Normally rapidly degraded by intracellular enzymes, inclusion of a β-hairpin â??protectideâ?? on the N-terminus of a peptide substrate increases reporter stability inside the cell. Effective transport of these peptide reporters into intact cells has presented several challenges to researchers. For example, one method of transport is microinjection with a needle, but this process can damage the cell. However, a cell penetrating peptide (CPP) sequence can overcome these limitations. This research presents a library of short peptide sequences that use a β-hairpin motif as both a protectide and a CPP. Members of this peptide library demonstrated the ability to rapidly penetrate the cell membrane of intact HeLa cells independent of incubation time and temperatures assessed by both fluorometry and fluorescent microscopy. β-hairpin permeability efficiency was compared to commercially available CPPs [e.g., TAT and Arg(9)] and a negative control peptide known to not penetrate intact cells. Two peptides named RWRWR [Ac-RWVRVpGO(FAM)WIRQ-NH₂] and OWRWR [Ac-OWVRVpGO(FAM)WIRQ-NH₂] were discovered to exhibit the highest permeability efficiency. Peptide stability was demonstrated using a degradation assay coupled with reverse phase high performance liquid chromatography (RP-HPLC). The commercial peptides , while superior in cellular uptake efficiency, displayed showed reduced cellular stability. Additional members of the peptide library were used to show that cellular uptake was not dependent on the secondary structure of the β-hairpins, but the lack of the secondary structure showed decreased cellular stability. Additionally, orthinine-rich members of the peptide library have been previously discovered to act as primary degrons, resulting in in vitro ubiquitination. Here, it was determined that these peptides could not only gain entry to intact HeLa cells, but could also be rapidly ubiquitinated. This combination of attributes offers significant promise for these β-hairpin protectides to serve as next generation biosensors for members of the ubiquitin proteasome system.