Arginine-Rich Intracellular Delivery Peptides Synchronously Deliver Covalently and Noncovalently Linked Proteins into Plant Cells

Abstract

Protein transduction domains (PTDs) are small peptides with a high content of basic amino acids, and they are responsible for cellular uptake. Many PTDs, including arginine-rich intracellular delivery (AID) peptides, have been shown to transport macromolecules across membranes and into cells. In this study, we demonstrated for the first time that AID peptides could rapidly and efficiently deliver proteins into plant cells in both covalent and noncovalent protein transductions (CNPT) simultaneously. The optimal molecular ratio between an AID peptide carrier and cargo in CNPT was about 3:1. Fluorescence resonance energy transfer (FRET) analysis revealed protein-protein interactions between AID peptide carriers and cargos after CNPT in cells. The possible mechanisms of AID peptides-mediated cellular entry might involve a combination of multiple internalization pathways. Therefore, applications by AID peptide-mediated CNPT may provide a simple and direct transport strategy for delivering two proteins in agricultural systems.

Intracellular delivery of quantum dots mediated by a histidine- and arginine-rich HR9 cell-penetrating peptide through the direct membrane translocation mechanism

“…Ruan et al found that macropinocytosis which involved actin filaments and microtubules was the major route for CPP-QDs uptake and intracellular transport [27]. In our recent studies, CPPs were found to enter into cells in covalent and noncovalent protein transduction (CNPT) manners that involved multiple pathways [34,35]. All of these studies support the notion that both cargoes and conjugation strategies influence the route of cellular internalization of CPPs.…”

“…PB, a counter-anion of positively charged CPPs, also increases hydrophobicity and accelerates translocations across membranes [39]. We found that R9-GFP, a covalent CPP fusion protein [34,35], exhibited increased transduction in treatments with both R9-GFP and PB (Fig. 8G).…”

Arginine-rich cell-penetrating peptides deliver gene into living human cells

“…CPPs have been shown to be very attractive and noncytotoxic candidates for the delivery of therapeutic macromolecules, such as proteins and nucleic acids (El-Sayed et al, 2009;Li et al, 2010;Liu et al, 2007;Lu et al, 2010;Margus et al, 2012;Nakase et al, in press, 2012;Raagel et al, 2010;van den Berg and Dowdy, 2011). The safety of most CPPs has been confirmed in a detailed metabolic analysis (Kilk et al, 2009).…”

Gene transport and expression by arginine-rich cell-penetrating peptides in Paramecium

“…Insulin-FITC (Sigma-Aldrich, St. Louis, MO, USA) was applied as a non-CPP reference as previously described . GFP was purified from Escherichia coli transformed with the pGFP1 plasmid (Lu et al, 2010) as previously described (Chang et al, 2005a).…”

“…In recent years, we have demonstrated that a CPP (synthetic nonaarginine; SR9) is able to transport fluorescent protein cargos in a noncovalent manner into living animal and plant cells Hou et al, 2007;Hu et al, 2009;Lu et al, 2010;Wang et al, 2006) and into other organisms including cyanobacteria, bacteria, archaea and yeasts (Liu et al, 2008). Moreover, this SR9 could deliver DNA Lee et al, 2011), RNA (Wang et al, 2007) or nanoparticles (Liu et al, 2010a(Liu et al, ,b, 2011Xu et al, 2010) into living cells noncovalently.…”