Abstract
Cell-penetrating peptides (CPPs) have gained attention as promising tools to enable the delivery of various molecules in a non-invasive manner. Among the CPPs, TAT and poly-arginine have been extensively utilized in numerous studies for the delivery of functional proteins, peptides, and macromolecules to analyze cellular signaling. However, the molecular mechanisms of cellular entry remain largely unknown. Here, we applied siRNA library screening to identify the regulatory genes for the cellular entry of poly-arginine peptide based on microscopic observation of the entry of fluorescent peptides in siRNA-treated cells. In this screening, we identified the cell membrane gene SLC4A4 and the trafficking regulator gene COPA, which also plays an important role in early endosome maturation. These results demonstrated that cellular entry of poly-arginine requires at least two different steps, probably binding on the cell surface and endosomal entry. The identification of genes for cellular entry of poly-arginine provides insights into its mechanisms and should further aid in the development of highly efficient cell-penetrating peptides.
Highlights
The cell membrane is the barrier that separates the inside and outside of cells, and highly hydrophilic substances such as proteins, peptides, and nucleic acids are not usually transmitted through the cell membrane
Negatively charged heparan sulfate proteoglycans (HS) on the cell surface play an important role in binding to cationic Cell-penetrating peptides (CPPs), and cellular uptake occurs by the processes of endocytosis or macropinocytosis [15,16,17]
The fluorescence intensity of HeLa cells was measured by MetaMorph (Olympus, Tokyo, Japan), which showed that COPA, SLC4A4, ATP8B3, and CX40.1 siRNAs reduced the uptake of FITC-9R compared with scrambled siRNA (Fig. 1B)
Summary
The cell membrane is the barrier that separates the inside and outside of cells, and highly hydrophilic substances such as proteins, peptides, and nucleic acids are not usually transmitted through the cell membrane. Using cell-penetrating peptides (CPP) as vectors, methods have been developed for introducing membraneimpermeable molecules into cells Among these CPPs, the cationic TAT peptide derived from human immunodeficiency virus type 1 (HIV-1) [1,2,3] and the arginine-rich peptide (9R–11R) are the most frequently used [4,5,6]. The molecular mechanisms of CPP cellular entry remain largely unknown To elucidate these molecular mechanisms, the identification of genes involved in the cellular uptake of cationic peptides is required in order to enhance their efficiency. We identified potential regulator genes involved in the cellular entry of 9R peptide using a genome-wide siRNA library containing 991 human membrane-associated genes
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