Gen Tanaka

Cell-surface Accumulation of Flock House Virus-derived Peptide Leads to Efficient Internalization via Macropinocytosis

Arginine-rich cell-penetrating peptides (CPPs), including human immunodeficiency virus type 1 (HIV-1) Tat (48-60) and oligoarginines, have been applied as carriers for delivery of cargo molecules, because of their capacity to internalize into cells and penetrate biological membranes. Despite the fact that they have been extensively studied, the factors required for the efficient internalization of CPPs are still unclear. In this report, we evaluated the internalization efficiencies of seven CPPs derived from DNA/RNA-binding peptides, and discovered that a peptide derived from the flock house virus (FHV) coat protein was internalized most efficiently into Chinese hamster ovary (CHO-K1), HeLa, and Jurkat cells. Comparison of the factors facilitating the internalization with those of the Tat peptide revealed that the FHV peptide induces macropinocytosis much more efficiently than the Tat peptide, which leads to its high cellular uptake efficiency. Additionally, the strong adsorption of the FHV peptide on cell membranes via glycosaminoglycans (GAGs) was shown to be a key factor for induction of macropinocytosis, and these steps were successfully monitored by live imaging of the peptide internalization into cells in relation to the actin organization. The remarkable methods of FHV peptide internalization thus highlighted the critical factors for internalizations of the arginine-rich CPPs.

CXCR4 Stimulates Macropinocytosis: Implications for Cellular Uptake of Arginine-Rich Cell-Penetrating Peptides and HIV

CXCR4 is a coreceptor of HIV-1 infection in host cells. Through a photocrosslinking study to identify receptors involved in internalization of oligoarginine cell-penetrating peptides (CPPs), we found that CXCR4 serves as a receptor that stimulates macropinocytic uptake of the arginine 12-mer peptide (R12) but not of the 8-mer. We also found that stimulating CXCR4 with its intrinsic ligands, stromal cell-derived factor 1α and HIV-1 envelope glycoprotein 120, induced macropinocytosis. R12 had activity to prevent viral infection for HIV-1(IIIB), a subtype of HIV-1 that uses CXCR4 as a coreceptor for entry into susceptible cells, whereas the addition of a macropinocytosis inhibitor, dimethylamiloride, resulted in enhancement of viral infection. The present study shows that CXCR4 triggers macropinocytosis, which may have implications for the cellular uptake of oligoarginine CPPs and internalization of HIV.

Effect of the attachment of a penetration accelerating sequence and the influence of hydrophobicity on octaarginine-mediated intracellular delivery.

Arginine-rich cell-penetrating peptides (CPPs), including oligoarginine peptides, have been widely used as a tool for intracellular delivery of various molecules with low membrane permeability. We previously reported the enhanced cytosolic entry of arginine-rich CPPs by the attachment of a short peptide segment, the penetration accelerating sequence (Pas). In this study, the importance of hydrophobic sequences, especially phenylalanine residues, in the Pas segment was demonstrated for this enhanced translocation through cell membranes. The advantage of using Pas for intracellular delivery was particularly marked for delivering cargoes with a relatively small molecular weight, such as bioactive peptides. In addition, the results of this study indicate the important roles that the total hydrophobicity of the PasR8 conjugates play in cytosolic translocation and the eventual bioactivity thus attained.

Signal Transduction Using an Artificial Receptor System that Undergoes Dimerization Upon Addition of a Bivalent Leucine‐Zipper Ligand

Dimerization and clustering of biological receptors on the plasma membrane lead to activation and subsequent signal transduction. If this dimerization event could be artificially controlled, it would be a valuable tool for studying signal transduction and modulating cellular functions. Previous studies have applied antibodies that recognize pre-incorporated tags on cell-surface receptors to stimulate receptor dimerization. Dimerization of receptors expressing a single-chain antibody against fluorescein by adding fluorescein-modified albumins were also reported. 4] Although antibodies are specific and have high ligand-binding affinities, their bulky structures may limit the design of ligand-receptor recognition systems. Other approaches caused intracellular cross-linking using FK506and FKBP-tagged intracellular signaling domains of receptors to form dimers when FKBP binds FK506. These approaches often employed multiple intracellular FKBP domains, which could lead to the assembly of more than two intracellular domains upon the addition of dimeric FK506. Possible steric interference of FKBP multimers might induce interaction of intracellular domains different from those of the wild-type receptors. Herein, we propose a novel approach to controlled receptor activation that employs a leucine-zipper coiled-coil as a recognition element to stimulate dimerization (Figure 1). This approach provides flexibility in the selection of the different combinations of leucine zippers, as recognition elements, and the linkers tethering them to the receptors being studied. To demonstrate the feasibility of this approach, we used a heterodimeric coiled-coil developed by Hodges and coworkers (E/K coil). This recognition motif has already been used to fluorescently label cell-surface proteins. 8] Peptide probes K3 (= (KIAALKE)3) and K4 (= (KIAALKE)4), labeled with a fluorophore, specifically stained the surfaceexposed tag sequence E3 (= (EIAALEK)3) attached to the N-terminus of the proteins. Recognition of these peptides is quick (< 1 min) with a high affinity (Kd = 64 nm for K3 and 6 nm for K4), which should also allow for efficient dimerization of receptor proteins. Considering that the K4 peptide showed a higher affinity to E3 than K3, we designed a system using bivalent K4 ligands to bring together cell-surface receptors bearing the E3 tag (Figure 1). One recent study employed an extracellular leucine-zipper dimerization domain of epidermal growth factor receptor (EGFR). This previous work used a constitutively dimerized leucinezipper–EGFR fusion to analyze the mechanism of an intracellular EGFR activating factor. On the other hand, our approach could increase the flexibility in selection of combinations of leucine zippers as recognition elements and in the linkers tethering them to the receptors. In addition, this method should provide an additional level of control by allowing a triggered dimerization event in biochemical and cell-biological experiments. EGFR is a representative receptor tyrosine kinase (RTK) that regulates proliferation, motility, and survival in normal cells, and is also implicated in many human cancers. Binding of EGF promotes dimerization of the EGFR, which causes phosphorylation of the cytoplasmic domain and activation of downstream signaling pathways. 10, 12, 13] The extracellular region of EGFR contains four subdomains (domains I–IV). 12] We designed an EGFR receptor lacking domains I–III and a part of domain IV (which are responsible for binding to EGF and dimerization) but included hemagglutinin A (HA) and E3 tags (E3-EGFR; tags are for the Figure 1. Scheme of artificial EGFR activation by a helical peptide through coiled-coil formation.

Molecular interplays involved in the cellular uptake of octaarginine on cell surfaces and the importance of syndecan-4 cytoplasmic V domain for the activation of protein kinase Cα.

Arginine-rich cell-penetrating peptides (CPPs) are promising carriers for the intracellular delivery of various bioactive molecules. However, many ambiguities remain about the molecular interplays on cell surfaces that ultimately lead to endocytic uptake of CPPs. By treatment of cells with octaarginine (R8), enhanced clustering of syndecan-4 on plasma membranes and binding of protein kinase Cα (PKCα) to the cytoplasmic domain of syndecan-4 were observed; these events potentially lead to the macropinocytic uptake of R8. The cytoplasmic V domain of syndecan-4 made a significant contribution to the cellular uptake of R8, whereas the cytoplasmic C1 and C2 domains were not involved in the process.

Identification of cellular proteins interacting with octaarginine (R8) cell-penetrating peptide by photo-crosslinking

Octaarginine (R8) is a representative cell-penetrating peptide. Lanthionine synthetase component C-like protein 1 (LanCL1) was identified as a potential intracellular target of R8 by using a photo-crosslinking assay that utilized a phenyl-trifluoromethyl diazirine moiety and peptide mass fingerprinting. Increased cellular uptake of R8 by LanCL1-overexpressing cells was observed.

Expressed protein ligation for the preparation of fusion proteins with cell penetrating peptides for endotoxin removal and intracellular delivery

Expressed protein ligation (EPL) is a useful method for the native chemical ligation of proteins with other proteins or peptides. This study assessed the practicability of EPL in the preparation of fusion proteins of enhanced green fluorescent protein (EGFP) with chemically synthesized cell-penetrating peptides (CPPs) for intracellular delivery. Using intein-mediated purification with an affinity chitin-binding tag (IMPACT) system, the thioester of EGFP (EGFP-SR) was prepared. Optimization of the ligation of EGFP-SR with arginine 12-mer (R12) produced the fusion protein in high yield. The EPL procedure also allows the preparation of EGFP-R12 containing a low level of endotoxin (ET), via the satisfactory ET removal of EGFP-SR prior to ligation with the R12 peptide. Fusion proteins of EGFP with R12 and the d-isomer of R12 prepared by EPL showed similar levels of cellular uptake compared to the fusion protein directly expressed in Escherichiacoli.

Photoaffinity Labeling Methods to Explore Internalization Mechanisms of Arginine-Rich Cell-Penetrating Peptides

Peptides with membrane permeability have been used extensively as carriers for intracellular delivery of bioactive molecules, and endocytosis plays an important role in the cellular uptake of these peptides. Elucidating the mechanisms of uptake will contribute to the development of more sophisticated delivery systems. In this chapter, we introduce a novel approach for identifying receptors involved in the cellular uptake of oligoarginines, a representative class of cell-penetrating peptides (CPPs). Photoaffinity probes with oligoarginines together with trifluoromethyl diazirines as photoreactive moieties and biotin as an isolation tag were designed. We identified C-X-C chemokine receptor type 4 (CXCR4) as a receptor for dodecaarginine (R12) uptake via macropinocytosis, a typical uptake pathway for arginine-rich CPPs. Interestingly, CXCR4 does not play a significant role in the uptake of octaarginine (R8) and HIV-1 TAT peptides, suggesting that more than one form of macropinocytosis is involved in arginine-rich CPP uptake. The cytoplasmic protein LanCL1 was also identified because of its ability to promote R8 cellular uptake. A novel probe with a diazobenzene linker, which allowed chemoselective cleavage of the photocrosslinked proteins from affinity beads, facilitated isolation of the target proteins. Using this probe, syndecan-4 was identified as a receptor involved in R8 uptake via clathrin-mediated endocytosis.