Unique arginine array improves cytosolic localization of hydrocarbon-stapled peptides

Publication date: Available online 7 November 2017
Source:Bioorganic & Medicinal Chemistry
Author(s): Kim Quach, Jonathan LaRochelle, Xiao-Han Li, Elizabeth Rhoades, Alanna Schepartz
We have previously reported that miniature proteins containing a distinct array of 5 arginine residues on a folded α-helix–a penta-arg motif–traffic with high efficiency from endosomes into the cytosol and nucleus of mammalian cells. Here we evaluate whether a penta-arg motif can improve the intracellular trafficking of an otherwise impermeant hydrocarbon-stapled peptide, SAH-p53-4^Rho. We prepared a panel of SAH-p53-4^Rho variants containing penta-arg sequences with different spacings and axial arrangement and evaluated their overall uptake (as judged by flow cytometry) and their intracellular access (as determined by fluorescence correlation spectroscopy, FCS). One member of this panel reached the cytosol extremely well, matching the level achieved by SAH-p53-8^Rho, a previously reported and highly permeant hydrocarbon-stapled peptide. Notably, we found no relationship between cellular uptake as judged by flow cytometry and cytosolic access as determined by FCS. This result emphasizes the importance of accessing intracellular access directly via FCS or an analogous method and reiterates that overall uptake and endosomal release represent fundamentally different biological processes. To determine cytosolic and/or nuclear access, one must measure concentration directly using a quantitative tool such as FCS. Our results also suggest that optimal penetration of hydrocarbon-stapled peptides into the cell cytosol results when the penta-arg motif is located within more (as opposed to less) structured regions.

Graphical abstract

http://ift.tt/2yFfl3n