(147a) Rapid Uptake and Ubiquitination of Fluorescent Peptides into Intact Cancer Cells Using a β-Hairpin Sequence Motif | AIChE

(147a) Rapid Uptake and Ubiquitination of Fluorescent Peptides into Intact Cancer Cells Using a β-Hairpin Sequence Motif


Melvin, A. - Presenter, Louisiana State University
Safabakhsh, N., Louisiana State University
Anderson, J., Louisiana State University
Vaithiyanathan, M., Louisiana State University
Pappas, G., Louisiana State University
Pettigrew, J., Louisiana State University
Gauthier, T., Louisiana State University
Cell penetrating peptides (CPPs) have emerged as powerful tools for delivering bioactive cargoes, such as biosensors and chemotherapeutics, to cells. CPPs possess several advantages over competing techniques like microinjection, including their ease of synthesis, compatibility with high-throughput analysis techniques, and decreased effect on cellular viability; however, the application of CPPs may be limited as a result of their degradation by intracellular proteases. In this research, a library of short peptide sequences, termed â??protectidesâ??, were synthesized using solid phase peptide synthesis that exhibit β-hairpin motif. These short peptides exhibited a prominent secondary structure which resulted in increased stability under cytosolic conditions. The effect of incubation time, temperature, and concentration was correlated to cellular uptake efficiency and long-term stability in cell lysates. Interestingly, the ornithine-rich members of the peptide library were also found to act as ubiquitination sensors capable of in vitro ubiquitination in intact cells. All members of the β-hairpin peptide library demonstrated the ability to rapidly penetrate the cell membrane of intact HeLa cells, using both fluorometry and microscopy techniques, although to various extents. Peptide uptake was found to be both temperature and concentration dependent. The arginine-rich peptides named RWRWR [Ac-RWVRVpGO(FAM)WIRQNH2] and OWRWR [Ac-OWVRVpGO(FAM)WIRQ-NH2], were discovered to exhibit the highest permeability efficiency. The stability of the members of the peptide library under conditions similar to the cytosolic environment was verified using a degradation assay and reverse phase high performance liquid chromatography (RP-HPLC). The β-turn was confirmed using circular dichroism (CD). While commercial peptides TAT and Arg(9) demonstrated superior permeability efficiency, they also exhibited decreased intracellular stability compared to members of the protectide library. The scrambled versions of the peptides revealed that the secondary structure is not necessary for peptide internalization; however the unstructured variants showed increased cellular uptake in intact cells, but decreased stability in cell lysates. Finally, as a proof-of-concept, the protectide was incorporated into the N-terminus of an enzyme substrate to generate a long-lived, cell permeable fluorescent probe for detecting intracellular deubiquitinating enzyme activity in intact cancer cells.