Pathway for Polyarginine Entry into Mammalian Cells

Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin, United States
Biochemistry (Impact Factor: 3.02). 04/2004; 43(9):2438-44. DOI: 10.1021/bi035933x
Source: PubMed


Cationic peptides known as protein transduction domains (PTDs) provide a means to deliver molecules into mammalian cells. Here, nonaarginine (R(9)), the most efficacious of known PTDs, is used to elucidate the pathway for PTD internalization. Although R(9) is found in the cytosol as well as the nucleolus when cells are fixed, this peptide is observed only in the endocytic vesicles of live cells. Colocalization studies with vesicular markers confirm that PTDs are internalized by endocytosis rather than by crossing the plasma membrane. The inability of R(9) to enter living cells deficient in heparan sulfate (HS) suggests that binding to HS is necessary for PTD internalization. This finding is consistent with the high affinity of R(9) for heparin (K(d) = 109 nM). Finally, R(9) is shown to promote the leakage of liposomes but only at high peptide:lipid ratios. These and other data indicate that the PTD-mediated delivery of molecules into live mammalian cells involves (1) binding to cell surface HS, (2) uptake by endocytosis, (3) release upon HS degradation, and (4) leakage from endocytic vesicles.

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Available from: Stephen M Fuchs, Jul 11, 2014
    • "For example, one of the critical factors which may affect the uptake pathway is the concentration of the peptide used. At higher concentrations a perturbation or even a disruption of the membrane may occur, leading to a passive entry of the peptide [59]. At the peptide concentrations employed in this study (10 and 20 μM), that however are near to those found in the oral cavity in saliva, we could not detect any membrane damage as evidenced by the microscopy observations, cytotoxicity, and artificial vesicle assays, performed even at concentrations well above those used in the uptake studies. "
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    ABSTRACT: Saliva contains hundreds of small proline-rich peptides most of which derive from the post-translational and post-secretory processing of the acidic and basic salivary proline-rich proteins. Among these peptides we found that a 20 residues proline-rich peptide (p1932), commonly present in human saliva and patented for its antiviral activity, was internalized within cells of the oral mucosa. The cell-penetrating properties of p1932 have been studied in a primary gingival fibroblasts cell line and in a squamous cancer cell line, and compared to its retro-inverso form. We observed by mass-spectrometry, flow cytometry and confocal microscopy that both peptides were internalized in the two cell lines on a time scale of minutes, being the natural form more efficient than the retro-inverso one. The cytosolic localization was dependent on the cell type: both peptide forms were able to localize within nuclei of tumoral cells, but not in the nuclei of gingival fibroblasts. The uptake was shown to be dependent on the culture conditions used: peptide internalization was indeed effective in a complete medium than in a serum-free one allowing the hypothesis that the internalization could be dependent on the cell cycle. Both peptides were internalized likely by a lipid raft-mediated endocytosis mechanism as suggested by the reduced uptake in the presence of methyl-ß-cyclodextrin. These results suggest that the natural peptide may play a role within the cells of the oral mucosa after its secretion and subsequent internalization. Furthermore, lack of cytotoxicity of both peptide forms, highlights their possible application as novel drug delivery agents. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta 08/2015; DOI:10.1016/j.bbamem.2015.08.019 · 4.66 Impact Factor
    • "This impedes the development of these peptides as effective carriers of nucleic acid therapeutics. Negatively charged cell surface molecules, particularly the proteoglycans, have been implicated in the literature as possible partners in the entry process of cationic cell penetrating peptides [9] [10] [11]. However, their role in the entry (and the subsequent intracellular events) of arginine-rich peptides complexed with cargos is ambiguous. "
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    Biochimica et Biophysica Acta (BBA) - Biomembranes 01/2015; 1848(4). DOI:10.1016/j.bbamem.2015.01.012 · 3.84 Impact Factor
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    • "Despite their clinical potential, the use of CPPs has been hampered by the fact that their internalized cargos remain mostly trapped in endocytic vesicles, leading to their subsequent degradation or recycling out of cells [1] [3]. Endosomal entrapment leads to a substantial reduction (to less than 1%) in the amount of CPP-delivered cargos reaching targets residing in the cytosol or other subcellular compartments [4] [5] [6] [7] [8]. "
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    ABSTRACT: The use of cell-penetrating peptides (CPPs), such as polyarginine, has been shown to facilitate the import of drugs and other cargos into cells. However, a major obstacle limiting their use as delivery agents is their entrapment following internalization into endocytic vesicles, leading to either their recycling out of cells or their degradation in lysosomes. To address this challenge, we fused a CPP sequence to the translocation domain of Pseudomonas aeruginosa exotoxin A (ETA) to facilitate the endosomal escape of imported CPP-containing protein constructs. Specifically, a fusion protein incorporating ten arginines linked to residues 253 to 412 of ETA (ETA(253-412)) was tested for its ability to effectively route a protein cargo (enhanced green fluorescent protein, eGFP) to the cytosol of cells. Using flow cytometry and fluorescence live-cell imaging, we observed a 5-fold improvement of cellular uptake as well as a 40-fold increase in cytosolic delivery of the CPP-ETA(253-412)-eGFP construct in relation to CPP-eGFP. Furthermore, analysis of intracellular routing events indicated that the incorporation of ETA(253-412) within the CPP-containing protein fusion construct avoided lysosomal degradation by re-directing the construct from early endosomes to the ER lumen and finally to the cytosol. Studies using inhibitors of vesicular transport confirmed that the ER lumen is a key compartment reached by the CPP-ETA(253-412)-eGFP construct before accessing the cytosol. Together, these findings suggest that incorporating a CPP motif and the ETA translocation domain into protein constructs can facilitate their cytosolic delivery.
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