Improved Assays for Determining the Cytosolic Access of Peptides, Proteins, and Their Mimetics

Abstract

Proteins and other macromolecules that cross biological membranes have great potential as tools for research and next-generation therapeutics. Here we describe two assays that effectively quantify the cytosolic localization of a number of previously reported peptides and protein domains. One assay, which we call GIGI (Glucocorticoid-Induced eGFP Induction), is an amplified assay that informs on relative cytosolic access without need for sophisticated imaging equipment or adherent cells. The second, GIGT (Glucocorticoid-Induced eGFP Translocation), is a non-amplified assay that informs on relative cytosolic access and exploits sophisticated imaging equipment to facilitate high-content screens in live cells. Each assay was employed to quantify the cytosolic delivery of several canonical “cell permeable peptides”, as well as more recently reported minimally cationic miniature proteins and zinc finger nuclease domains. Our results show definitively that both overall charge as well as charge distribution influence cytosolic access, and that small protein domains containing a discrete, helical, penta-arg motif can dramatically improve the cytosolic delivery of small folded proteins such as zinc finger domains. We anticipate that the assays described herein will prove useful to explore and discover the fundamental physicochemical and genetic properties that influence both the uptake and endosomal release of peptidic molecules and their mimetics.

Getting Across the Cell Membrane: An Overview for Small Molecules, Peptides, and Proteins

“…For example, peptides have been conjugated to dexamethasone (Dex) derivatives, which bind to transiently expressed gluococorticoid receptor (GR)-fusion proteins in the cytosol to induce a reporter [28] or alter its localization [29]. It should be noted that reporter gene expression inherently amplifies the signal through multiple rounds of transcription and translation [29]. In cases where the biological activity of the pay-load is reported, certain payloads can generate the measured macroscopic effect with fewer numbers.…”

Mechanism Matters: A Taxonomy of Cell Penetrating Peptides

“…The convenience of fluorescent dyes as “cargo” has caused much of the field to focus, almost exclusively, on the discovery of peptides that can deliver dyes, instead of useful cargoes. The field will be better served by diversified approaches, such as some labs are pursuing[96], for detecting and quantitating the delivery of useful cargoes to cells.…”

Discovery and Characterization of a Peptide That Enhances Endosomal Escape of Delivered Proteins in Vitro and in Vivo

“…After 30 min, cells were washed twice with 200 µL of HEPES−Krebs−Ringer's (HKR) buffer (140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES at pH 7.4), after which 100 µL of HKR buffer was overlaid onto the cells and images were acquired on a Zeiss Axiovert 200M epifluorescence microscope outfitted with Ziess AxiocammRM camera and an EXFO-Excite series 120 Hg arc lamp. The translocation ratio (the ratio of median GFP intensity in the nuclear and surrounding regions) for individual cells was measured using CellProfiler® as described 36 . To examine the effect of endocytosis inhibitors, HeLa cells were pretreated for 30 min with DMEM (without phenol red) containing inhibitors (80 µM Dynasore, 5 mM MBCD, 50 µM EIPA, 200 nM bafilomycin or 200 nM wortmannin) at 37 °C for 30 min before incubation with Dex or Dex-protein conjugates.…”