- Email: [email protected]
Stimulated endocytosis in penetratin uptake: Effect of arginine and lysine
Biochemical and Biophysical Research Communications 371 (2008) 621–625
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Stimulated endocytosis in penetratin uptake: Effect of arginine and lysine Helene L. Åmand, Kristina Fant, Bengt Nordén, Elin K. Esbjörner * Department of Chemical and Biological Engineering/Physical Chemistry, Chalmers University of Technology, Kemivägen 10, S-412 96 Gothenburg, Sweden
a r t i c l e
i n f o
Article history: Received 19 March 2008 Available online 13 May 2008
Keywords: Cell-penetrating peptide Penetratin Stimulated endocytosis Macropinocytosis CHO-K1 Uptake Arginine Lysine
a b s t r a c t Cell-penetrating peptides can deliver macromolecular cargo into cells and show promise as vectors for intracellular drug delivery. Internalization occurs predominantly via endocytosis, but the exact uptake mechanisms are not fully understood. We show quantitatively how penetratin, a 16-residue cationic peptide, stimulates fluid-phase endocytosis and triggers its own uptake into Chinese hamster ovarian cells, using a 70 kDa dextran to indicate macropinocytosis. The total cellular endocytotic rate is significantly less affected and we therefore propose up-regulation of macropinocytosis to occur at the expense of other types of endocytosis. By comparing penetratin to its analogs PenArg and PenLys, enriched in arginines and lysines, respectively, we show how these side-chains contribute to uptake efficiency. The degree of peptide and dextran uptake follows similar patterns regarding peptide concentration and arginine/ lysine content (PenArg > penetratin > PenLys), indicating that a high content of arginines is beneficial but not necessary for stimulating endocytosis. Ó 2008 Elsevier Inc. All rights reserved.
Cell-penetrating peptides (CPPs) have emerged as promising vectors for intracellular delivery of cargos such as oligonucleotides, proteins, plasmid-DNA, liposomes, and also nanoparticles [1]. Despite their well-demonstrated ability to efficiently internalize macromolecular cargos, the internalization mechanisms are still relatively poorly understood. Early studies proposed non-endocytotic, receptor- and transporter-independent mechanisms and efforts were put into mimicking peptide entry in protein-free membrane model systems [2–8]. However, it has also been shown that some early results were exaggerating uptake efficacy due to false redistribution of plasma-membrane associated peptide during cell fixation, routinely used prior to imaging [9,10]. Re-evaluation of this field using live cells has resulted in new views of uptake mechanisms. Even though the concept of energy-independent internalization pathways still holds for some arginine-rich CPPs [11–15], endocytosis is nowadays commonly recognized as major pathway for internalization of CPP-cargo constructs [16]. This has however not facilitated the understanding of how CPPs enter cells. Several conceptually diverse endocytotic pathways have been proposed [1], and each CPP can likely use several pathways in parallel. It has been suggested that arginines have unique ‘‘magic” uptake-promoting properties compared to other positively charged residues [12,13,17–19] because its head-group (guanidinium) can form particularly stable bidentate ion pairs with, for example, phosphates or sulfates at the membrane surface [17,20,21]. This
* Corresponding author. Fax: +46 0 31 772 3858. E-mail address: [email protected] (E.K. Esbjörner). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.039
property has mainly been associated with non-endocytotic CPP internalization and direct membrane penetration has been suggested to occur via charge-neutralizing ion-pairing with hydrophobic counter-ions at the membrane interface [22,23]. We will here extend the comparison of arginines and lysines to investigate how these residues affect endocytotic uptake and will also explore to what extent CPPs trigger their own internalization by stimulating endocytosis. Qualitative indications of stimulated macropinocytosis, a lipid-raft-dependent and receptor-independent form of fluid-phase endocytosis [24], have been implicated for the CPPs Tat and oligoarginines [25,26]. This study will build on the classic 16 residue basic and amphipathic CPP penetratin, derived from the Antennapedia homeodomain in Drosophila [27,28], and on two analogs, PenArg and PenLys (see Table 1 for peptide sequences), designed by us to systematically explore the impact of arginines and lysines on CPP characteristics [13,23,29,30].
Materials and methods Materials. Fluorescein isothiocyanate (FITC) labelled peptides (>80% purity) were purchased from NeoMPS (San Diego). The dye was conjugated to the N-terminus via a gamma-amino butyric acid (GABA) linker. Texas red-labelled 70 kDa neutral dextran (TR-dextran) and FM 4-64 were from Invitrogen, and cell culture reagents from PAA Laboratories. Chinese hamster ovarian cells (CHO-K1) were a kind gift from Prof. Ülo Langel, Stockholm University. Cell culture. CHO-K1 cells were cultured in HAM’s F12 medium supplemented with fetal calf serum (10%) and L-glutamine (2 mM) at 37 °C and 5% CO2. The cells were trypsinized every 3–4 days and seeded at a density of 7500 cells/cm2. For quantitative uptake experiments cells were seeded in 12-well plates
622
H.L. Åmand et al. / Biochemical and Biophysical Research Communications 371 (2008) 621–625
Table 1 Peptide sequences Peptide
Sequence
Penetratin PenArg PenLys
FITC-GABA-RQIK IWFQ NRRM KWKK-NH2 FITC-GABA-RQIR IWFQ NRRM RWRR-NH2 FITC-GABA-KQIK IWFQ NKKM KWKK-NH2
Arginines are marked in bold and lysines are underlined.
(11000 cells/cm2) and cultured to 90–100% confluence. For confocal imaging, cells were seeded on round coverslips at a density of 11000 cells/cm2 and cultured for 2 days. Quantitative uptake. Cells were rinsed with serum-free HAM’s F12 and incubated with appropriate reagents in serum-free HAM’s F12 for 1 h (1, 5 or 10 lM peptide and/or 100 lg/ml TR-dextran or 5 lg/ml FM 4-64). The cells were rinsed twice with Hepes buffered saline (20 mM HEPES, 150 mM NaCl, pH 7.4) supplemented with 100 lg/ml heparin (10 times the concentration required to block uptake of the similarly charged CPP Tat [26]) to remove externally bound peptide. The cells were collected by trypsination (10 min) followed by centrifugation (5 min, 5000g, 4 °C) and thereafter lysed (0.1 M NaOH, on ice, 1 h). Cell lysates were transferred to black 96-well plates for fluorimetric quantification of internalized CPP and endocytosis marker. A FluoStar Optima Plate Reader (BMG Labtechnologies, Germany) with excitation/emission filter settings at 485/520 nm for FITC-labelled peptides and at 544/590 nm for TR-dextran and FM 4-64 was used. The total protein concentration in each sample was determined using a protein assay kit from BioRad. The protocol essentially follows reference [31]. All experiments were repeated at least three times and always performed in triplicate. The uptake levels are reported as the arithmetic mean of all samples, whereas the error bars represent the average maximum and minimum data spread. All data were corrected for background contributions by subtracting appropriate blanks and normalized against the total protein concentration in each sample. Confocal imaging. Cells for confocal imaging were rinsed with serum-free HAM’s F12 and incubated for 1 h at 37 °C with appropriate reagents. Prior to imaging cells were washed again and the coverslips were mounted with fresh media in a solution chamber. A confocal laser scanning microscopy system (Leica TCS SP2 RS, Wetzlar, Germany), with a PL APO 63/1.32 objective, was used for acquisition of confocal fluorescence images. The 488 nm line of an argon laser was used for excitation of FITC and the 543 and 594 nm lines of a helium–neon laser were used for excitation of FM 4-64 and TR-dextran, respectively. Images were acquired in sequential mode to avoid bleed-through between the photomultiplier tubes. Detector settings (photomultiplier high tension) and contrast were optimized for each sample.
Results Quantification of CPP uptake Fig. 1 shows the uptake of FITC-labelled PenLys, penetratin, and PenArg in live CHO-K1 cells obtained after 1 h incubation at 37 °C. Uptake rate is in the order PenArg > penetratin > PenLys, indicating
Fig. 1. Uptake of penetratin peptides after 1 h incubation at 37 °C as a function of peptide concentration. Column height represents the average of nine independent experiments, performed in triplicate (N = 9, n = 3). Error bars indicate the average maximum and minimum deviation in each experiment (see text for further details).
that arginine-rich peptides are more efficiently internalized than lysine-rich ones. PenLys is however internalized in CHO-K1 cells (see also Fig. 2A), a finding that disputes previous observations and will be further considered in the Discussion. The uptake efficiency is roughly linearly dependent on peptide concentration and uptake is not saturable within this concentration regime. Care was taken to avoid measuring ‘‘false” fluorescence from externally bound peptide: cells were rinsed twice with HBS buffer supplemented with excess amounts of negatively charged heparin (see Materials and methods). Thereafter, cells were subjected to trypsination, a procedure that should remove any residual bound peptide and also help in degrading heparin-sequestered peptide, preventing resorption to the cell surface during the centrifugation step. Confocal imaging To confirm the intracellular location of peptide (vesicular versus cytoplasmic), confocal laser images were obtained on CHO-K1 cells incubated with CPPs and/or endocytosis markers for 1 h (Fig. 2). Fig. 2A shows peptide uptake and co-localization with the nonspecific marker of endocytosis FM 4-64. The detector settings and contrast were adjusted for best visualization of each image and the intensities are therefore not comparable. Intracellular fluorescence is observed for all three peptides and the punctuate pattern strongly indicates vesicular localization within the cell, thus confirming endocytotic uptake. Substantial, but not perfect, colocalization with FM 4-64 is observed (orange/yellow color), indicating that not all endocytotic vesicles contain peptide. Fig. 2B shows uptake of TR-dextran, a fluid-phase endocytosis marker used to indicate macropinocytosis, in absence and presence of peptide. TR-dextran is negligibly internalized in absence of peptide, whereas even the least well-internalized peptide (PenLys) markedly increases its uptake. TR-dextran co-localizes well with peptide, but some vesicular structures containing only peptide are also observed. Fig. 2C shows uptake of PenArg at an elevated concentration (10 lM). Both diffuse cytoplasmic and punctuate fluorescence are observed, but the transmitted image indicates that these cells have severely compromised morphology. This micrograph was included to demonstrate that PenArg is toxic at elevated concentrations. Such behavior was not observed for penetratin or PenLys under any conditions (data not shown). Quantification of stimulated endocytosis To assess to what extent penetratin peptides can stimulate endocytosis, quantitative measurements were performed where peptide was co-incubated with FM 4-64 or TR-dextran. FM 4-64 is a non-specific endocytosis marker that fluoresces only when bound to membranes. It cannot translocate across the plasma membrane and is only internalized via endocytosis. TR-dextran is used as marker of fluid-phase endocytosis. Dextrans of this size (70 kDa) are preferentially internalized by macropinocytosis compared to micropinocytotic mechanisms including clathrin-, caveolae-, and receptor-mediated endocytosis [32]. Fig. 3 shows intrinsic cell uptake of FM 4-64 (control) and uptake in presence of peptide. No statistically significant increase in uptake of FM 4-64 can be detected for PenLys or for penetratin and PenArg applied at 1 lM concentration. Some increased uptake is observed for the highest penetratin concentration and for PenArg applied at 5 and 10 lM. For PenArg, 10 lM, we cannot exclude that the exaggerated internalization is not due to peptide-mediated permeabilization of the plasma membrane (see Fig. 2C). Fig. 4 shows uptake of TR-dextran in presence of peptide. TR-dextran is not efficiently internalized in CHO-K1 cells under normal conditions (see also Fig. 2B). In presence of peptide, uptake is markedly increased, even at 1 lM peptide concentration. The amount of internalized TR-dextran
H.L. Åmand et al. / Biochemical and Biophysical Research Communications 371 (2008) 621–625
623
Fig. 2. Confocal images of live CHO-K1 cells incubated with peptide and/or endocytosis markers. (A) Uptake and co-localization (yellow indicates very good overlap) of FITClabelled peptide (green) and non-specific endocytosis marker FM 4-64 (red). The peptide concentration was 5 lM and the FM 4-64 concentration was 5 lg/ml. (B) Uptake of TR-dextran (50 lg/ml) in absence and presence of peptide (5 lM PenLys). (C) Uptake and distribution of PenArg applied at 10 lM concentration. Note the compromised cell morphology. Images were acquired after 1 h incubation at 37 °C. The image contrast and the gain (high tension over the photomultiplier tube detectors) have been optimized for each micrograph and the intensities in each image are thus not comparable.
Fig. 3. Internalization of the non-specific endocytosis marker FM 4-64 in absence (control) and presence of penetratin peptides after 1 h incubation at 37 °C. The FM 4-64 concentration was 5 lg/ml. Column heights represents the average of three independent experiments, performed in triplicate (N = 3, n = 3). Error bars represent the average maximum and minimum deviation in each experiment (see text for details).
Fig. 4. Internalization of fluid-phase endocytosis marker TR-dextran in absence (control) and presence of penetratin peptides. The cells were incubated for 1 h at 37 °C. The TR-dextran concentration was 100 lg/ml. Column heights represents the average of three independent experiments, performed in triplicate (N = 3, n = 3). Error bars represent the average maximum and minimum deviation in each experiment (see text for details).
Discussion increases with peptide concentration and is more efficient for PenArg than for penetratin and PenLys. Control experiments verified that neither TR-dextran nor FM 4-64 affected the degree of peptide internalization (data not shown).
Despite the fact that almost 20 years have elapsed since the idea of cell-penetrating peptides as transporters emerged, our understanding of how cell-penetrating peptides like penetratin, Tat, or oligoarginine, enter cells is still elusive from several aspects (see
624
H.L. Åmand et al. / Biochemical and Biophysical Research Communications 371 (2008) 621–625
Introduction). Here we shed some light on the CPP uptake mechanisms by demonstrating how penetratin triggers its own internalization by stimulating endocytosis and by quantifying the relative importance of arginines versus lysines for the efficiency of this process. Arginine-rich peptides have been argued to display superior cell-penetrating abilities compared to their lysine-rich counterparts, some reports even indicate a difference so extreme that whilst arginine-rich CPPs enter cells efficiently, the corresponding lysine-rich versions are completely unable to do so [13,17]. This study proves that both lysine-rich (PenLys) and arginine-rich (PenArg) versions of penetratin can enter CHO-K1 cells, albeit the former less efficiently (Fig. 1). This study is in conflict with some previous results, particularly those of a live cell confocal imaging study on PenLys [13], but our results are consistent with biophysical studies of peptide–membrane interaction in cell membrane mimetic systems [6,23,29] and also understandable from a physico-chemical perspective (vide infra). We have confirmed that PenLys cell-penetrating ability is not restricted to CHO-K1, but also occurs in at least two other cell lines: NIH-3T3 mouse fibroblasts and NG108-15 neuroblastoma/rat glioma cells (unpublished observations). The uptake efficiency of PenArg exceeds that of PenLys with about one order of magnitude. This difference can partly be explained by PenArg’s greater affinity for lipid membranes, but reported surface partition coefficients (PenArg 1900 M 1 and PenLys 600 M 1 for binding to lipid vesicles with 40% negative charge [30]) are not sufficiently different to entirely explain a 10fold difference in internalization. One may argue, based on previous suggestions, that negatively charged heparan sulfates are the primary cell surface binding sites for CPPs [33–35], that a lipid bilayer is not a suitable model for CPP cell interaction. However, since the negatively charged entity in HS is an oxoanion (SO4 ) just as the lipid phosphate (PO4 ) and since penetratin binds to lipid bilayers primarily by electrostatic interactions, we think it can be justified to assume that binding affinity for HS will fall in the same order for the penetratins. This is certainly the case for at least one other negatively charged polyelectrolyte, namely DNA (Åmand et al., in progress). Therefore, whilst CPP cell surface affinity in part explains uptake efficiency we believe that also other factors, such as ability to trigger cellular response, must be involved in order to fully explain the different uptake characteristics of penetratin, PenArg, and PenLys. Which these factors could be and how they relate to the chemistry of arginines and lysines remain to be explored. The peptide internalization observed in the quantitative uptake assay (Fig. 1) was verified to be mainly endocytotic (Fig. 2A). Not even PenArg, which has previously been indicated to use direct membrane penetration mechanisms [13], displays any significant diffuse cytoplasmic fluorescence except at the highest applied concentration (10 lM) (Fig. 2). Two previous reports have indicated that the CPPs Tat and octaarginine stimulate macropinocytosis [26,36] and Khalil et al. estimate roughly a 40% increase in macropinocytotic activity for their octaarginine–liposome constructs [36]. This study proves that also penetratin peptides trigger their own internalization by stimulating fluid-phase uptake of a large molecular weight marker (TR-dextran) used as indicator for macropinocytosis. Taking a step further, this study also quantifies to what extent this mechanism is up-regulated and shows that all three peptides increase uptake of TR-dextran significantly (up to ten times). PenLys, 1 lM, is the least effective agent but still doubles the uptake. The efficiency comes, as for peptide internalization, in the order PenArg > penetratin > PenLys suggesting that these two events are directly related. The co-localization of peptide and TR-dextran is however not perfect (Fig. 2B); some internalized vesicular structures contain only peptide, but the opposite is much
more infrequent, showing in accordance with previous reports [1] that penetratin is not exclusively internalized by one type of endocytosis. This raises an imperative question which we will attempt to answer: Does penetratin stimulate only its own uptake, i.e. will endocytosis occur only at sites where the peptide binds, or does cell surface interactions initiate signalling cascades that increase the endocytotic activity of the entire cell? Fig. 3 gives some information to this end, showing that the penetratin peptides have only marginal effects on the overall endocytosis rate (measured using a non-specific endocytosis marker) compared to the effect on the macropinocytotic activity. For 10 lM penetratin and 5 lM PenArg the amount of internalized FM 4-64 has barely doubled (compared to an 8–10 times increased TR-dextran uptake under similar conditions). At even lower penetratin or PenArg concentrations and for PenLys the change in endocytotic activity is negligible. Thus, we conclude that penetratin peptides predominantly stimulate their own uptake by initiating macropinocytotic fluid-phase uptake in CHO-K1. Macropinocytosis initiates with cell membrane ruffling and thus it is reasonable to conclude that penetratin stimulates this activity by its cell surface interactions. It appears as if up-regulation of macropinocytosis to some extent occurs at the expense of other endocytotic pathways since uptake of FM 4-64 is only marginally affected. Possibly the cell strives at keeping its membrane turnover rate relatively constant. In conclusion, this study evidences that penetratin peptides can trigger their own uptake by stimulating macropinocytosis upon binding to the cell surface of CHO-K1 cells. We also find indications for that up-regulation of this pathway occurs on the expense of other endocytotic mechanisms. Further, comparison of PenArg, penetratin and PenLys confirms that incorporation of additional arginines into a CPP increases its efficiency, but the results obtained for PenArg raise the question whether ‘‘better” will also mean ‘‘more toxic”. Support for an increased toxicity of argininerich CPPs, compared to penetratin, can be found in one of few comparative CPP toxicity studies [37] and possibly penetratin owes its efficient and non-toxic CPP properties to a balanced mix of arginines and lysines. Acknowledgments This work was supported by funding from the Swedish Cancer Society and from the European Commission (contracts 012967, 005204 and 037783). Prof. Langel at Department of Neurochemistry, Stockholm University, Sweden is acknowledged for the kind gift of the CHO-K1 cells. References [1] Ulo Langel Handbook of Cell-Penetrating Peptides, second ed., CRC Press, Taylor & Francis Group, Boca Raton, London, New York, 2007. [2] P.E. Thorén, D. Persson, M. Karlsson, B. Nordén, The antennapedia peptide penetratin translocates across lipid bilayers—the first direct observation, FEBS Lett. 482 (2000) 265–268. [3] E. Barany-Wallje, S. Keller, S. Serowy, S. Geibel, P. Pohl, M. Bienert, M. Dathe, A critical reassessment of penetratin translocation across lipid membranes, Biophys. J. 89 (2005) 2513–2521. [4] J. Björklund, H. Biverståhl, A. Gräslund, L. Mäler, P. Brzezinski, Real-time transmembrane translocation of penetratin driven by light-generated proton pumping, Biophys. J. 91 (2006) L29–L31. [5] M. Magzoub, A. Pramanik, A. Gräslund, Modeling the endosomal escape of cellpenetrating peptides: transmembrane pH gradient driven translocation across phospholipid bilayers, Biochemistry 44 (2005) 14890–14897. [6] D. Persson, P.E. Thorén, E.K. Esbjörner, M. Goksör, P. Lincoln, B. Nordén, Vesicle size-dependent translocation of penetratin analogs across lipid membranes, Biochim. Biophys. Acta 1665 (2004) 142–155. [7] D. Terrone, S.L. Sang, L. Roudaia, J.R. Silvius, Penetratin and related cellpenetrating cationic peptides can translocate across lipid bilayers in the presence of a transbilayer potential, Biochemistry 42 (2003) 13787–13799. [8] P.E. Thorén, D. Persson, E.K. Esbjörner, M. Goksör, P. Lincoln, B. Nordén, Membrane binding and translocation of cell-penetrating peptides, Biochemistry 43 (2004) 3471–3489.
H.L. Åmand et al. / Biochemical and Biophysical Research Communications 371 (2008) 621–625 [9] M. Lundberg, M. Johansson, Positively charged DNA-binding proteins cause apparent cell membrane translocation, Biochem. Biophys. Res. Commun. 291 (2002) 367–371. [10] J.P. Richard, K. Melikov, E. Vives, C. Ramos, B. Verbeure, M.J. Gait, L.V. Chernomordik, B. Lebleu, Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake, J. Biol. Chem. 278 (2003) 585–590. [11] A. Fischer, T. Oberholzer, P.L. Luisi, Giant vesicles as models to study the interactions between membranes and proteins, Biochim. Biophys. Acta 1467 (2000) 177–188. [12] D.J. Mitchell, D.T. Kim, L. Steinman, C.G. Fathman, J.B. Rothbard, Polyarginine enters cells more efficiently than other polycationic homopolymers, J. Pept. Res. 56 (2000) 318–325. [13] P.E. Thorén, D. Persson, P. Isakson, M. Goksör, A. Önfelt, B. Nordén, Uptake of analogs of penetratin, Tat(48–60) and oligoarginine in live cells, Biochem. Biophys. Res. Commun. 307 (2003) 100–107. [14] F. Duchardt, M. Fotin-Mleczek, H. Schwarz, R. Fischer, R. Brock, A comprehensive model for the cellular uptake of cationic cell-penetrating peptides, Traffic 8 (2007) 848–866. [15] M.M. Fretz, N.A. Penning, S. Al-Taei, S. Futaki, T. Takeuchi, I. Nakase, G. Storm, A.T. Jones, Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells, Biochem. J. 403 (2007) 335–342. [16] M. Fotin-Mleczek, R. Fischer, R. Brock, Endocytosis and cationic cellpenetrating peptides–a merger of concepts and methods, Curr. Pharm. Des. 11 (2005) 3613–3628. [17] J.B. Rothbard, T.C. Jessop, R.S. Lewis, B.A. Murray, P.A. Wender, Role of membrane potential and hydrogen bonding in the mechanism of translocation of guanidinium-rich peptides into cells, J. Am. Chem. Soc. 126 (2004) 9506– 9507. [18] M. Nishihara, F. Perret, T. Takeuchi, S. Futaki, A.N. Lazar, A.W. Coleman, N. Sakai, S. Matile, Arginine magic with new counterions up the sleeve, Org. Biomol. Chem. 3 (2005) 1659–1669. [19] S. Futaki, T. Suzuki, W. Ohashi, T. Yagami, S. Tanaka, K. Ueda, Y. Sugiura, Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery, J. Biol. Chem. 276 (2001) 5836–5840. [20] N. Sakai, S. Matile, Anion-mediated transfer of polyarginine across liquid and bilayer membranes, J. Am. Chem. Soc. 125 (2003) 14348–14356. [21] N. Sakai, T. Takeuchi, S. Futaki, S. Matile, Direct observation of anionmediated translocation of fluorescent oligoarginine carriers into and across bulk liquid and anionic bilayer membranes, Chembiochem. 6 (2005) 114–122. [22] J.B. Rothbard, T.C. Jessop, P.A. Wender, Adaptive translocation: the role of hydrogen bonding and membrane potential in the uptake of
[23]
[24] [25]
[26]
[27]
[28]
[29]
[30] [31] [32]
[33]
[34] [35] [36]
[37]
625
guanidinium-rich transporters into cells, Adv. Drug. Deliv. Rev. 57 (2005) 495–504. E.K. Esbjörner, P. Lincoln, B. Nordén, Counterion-mediated membrane penetration: cationic cell-penetrating peptides overcome Born energy barrier by ion-pairing with phospholipids, Biochim. Biophys. Acta 1768 (2007) 1550– 1558. S.D. Conner, S.L. Schmid, Regulated portals of entry into the cell, Nature 422 (2003) 37–44. A. Iwasa, H. Akita, I. Khalil, K. Kogure, S. Futaki, H. Harashima, Cellular uptake and subsequent intracellular trafficking of R8-liposomes introduced at low temperature, Biochim. Biophys. Acta 1758 (2006) 713–720. J.S. Wadia, R.V. Stan, S.F. Dowdy, Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis, Nat. Med. 10 (2004) 310–315. A. Joliot, C. Pernelle, H. Deagostini-Bazin, A. Prochiantz, Antennapedia homeobox peptide regulates neural morphogenesis, Proc. Natl. Acad. Sci. USA 88 (1991) 1864–1868. D. Derossi, A.H. Joliot, G. Chassaing, A. Prochiantz, The third helix of the Antennapedia homeodomain translocates through biological membranes, J. Biol. Chem. 269 (1994) 10444–10450. C.E. Caesar, E.K. Esbjörner, P. Lincoln, B. Nordén, Membrane interactions of cell-penetrating peptides probed by tryptophan fluorescence and dichroism techniques: correlations of structure to cellular uptake, Biochemistry 45 (2006) 7682–7692. D. Persson, P.E.G. Thorén, P. Lincoln, B. Nordén, Vesicle membrane interactions of penetratin analogues, Biochemistry 43 (2004) 11045–11055. T. Holm, H. Johansson, P. Lundberg, M. Pooga, M. Lindgren, Ü. Langel, Studying the uptake of cell-penetrating peptides, Nat. Protoc. 1 (2006) 1001–1005. N. Araki, M.T. Johnson, J.A. Swanson, A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages, J. Cell Biol. 135 (1996) 1249–1260. M. Tyagi, M. Rusnati, M. Presta, M. Giacca, Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans, J. Biol. Chem. 276 (2001) 3254– 3261. S.M. Fuchs, R.T. Raines, Pathway for polyarginine entry into mammalian cells, Biochemistry 43 (2004) 2438–2444. M. Belting, Heparan sulfate proteoglycan as a plasma membrane carrier, Trends Biochem. Sci. 28 (2003) 145–151. I.A. Khalil, K. Kogure, S. Futaki, H. Harashima, High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression, J. Biol. Chem. 281 (2006) 3544–3551. S.W. Jones, R. Christison, K. Bundell, C.J. Voyce, S.M. Brockbank, P. Newham, M.A. Lindsay, Characterisation of cell-penetrating peptide-mediated peptide delivery, Br. J. Pharmacol. 145 (2005) 1093–1102.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Stimulated endocytosis in penetratin uptake: Effect of arginine and lysine Helene L. Åmand, Kristina Fant, Bengt Nordén, Elin K. Esbjörner * Department of Chemical and Biological Engineering/Physical Chemistry, Chalmers University of Technology, Kemivägen 10, S-412 96 Gothenburg, Sweden
a r t i c l e
i n f o
Article history: Received 19 March 2008 Available online 13 May 2008
Keywords: Cell-penetrating peptide Penetratin Stimulated endocytosis Macropinocytosis CHO-K1 Uptake Arginine Lysine
a b s t r a c t Cell-penetrating peptides can deliver macromolecular cargo into cells and show promise as vectors for intracellular drug delivery. Internalization occurs predominantly via endocytosis, but the exact uptake mechanisms are not fully understood. We show quantitatively how penetratin, a 16-residue cationic peptide, stimulates fluid-phase endocytosis and triggers its own uptake into Chinese hamster ovarian cells, using a 70 kDa dextran to indicate macropinocytosis. The total cellular endocytotic rate is significantly less affected and we therefore propose up-regulation of macropinocytosis to occur at the expense of other types of endocytosis. By comparing penetratin to its analogs PenArg and PenLys, enriched in arginines and lysines, respectively, we show how these side-chains contribute to uptake efficiency. The degree of peptide and dextran uptake follows similar patterns regarding peptide concentration and arginine/ lysine content (PenArg > penetratin > PenLys), indicating that a high content of arginines is beneficial but not necessary for stimulating endocytosis. Ó 2008 Elsevier Inc. All rights reserved.
Cell-penetrating peptides (CPPs) have emerged as promising vectors for intracellular delivery of cargos such as oligonucleotides, proteins, plasmid-DNA, liposomes, and also nanoparticles [1]. Despite their well-demonstrated ability to efficiently internalize macromolecular cargos, the internalization mechanisms are still relatively poorly understood. Early studies proposed non-endocytotic, receptor- and transporter-independent mechanisms and efforts were put into mimicking peptide entry in protein-free membrane model systems [2–8]. However, it has also been shown that some early results were exaggerating uptake efficacy due to false redistribution of plasma-membrane associated peptide during cell fixation, routinely used prior to imaging [9,10]. Re-evaluation of this field using live cells has resulted in new views of uptake mechanisms. Even though the concept of energy-independent internalization pathways still holds for some arginine-rich CPPs [11–15], endocytosis is nowadays commonly recognized as major pathway for internalization of CPP-cargo constructs [16]. This has however not facilitated the understanding of how CPPs enter cells. Several conceptually diverse endocytotic pathways have been proposed [1], and each CPP can likely use several pathways in parallel. It has been suggested that arginines have unique ‘‘magic” uptake-promoting properties compared to other positively charged residues [12,13,17–19] because its head-group (guanidinium) can form particularly stable bidentate ion pairs with, for example, phosphates or sulfates at the membrane surface [17,20,21]. This
* Corresponding author. Fax: +46 0 31 772 3858. E-mail address: [email protected] (E.K. Esbjörner). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.039
property has mainly been associated with non-endocytotic CPP internalization and direct membrane penetration has been suggested to occur via charge-neutralizing ion-pairing with hydrophobic counter-ions at the membrane interface [22,23]. We will here extend the comparison of arginines and lysines to investigate how these residues affect endocytotic uptake and will also explore to what extent CPPs trigger their own internalization by stimulating endocytosis. Qualitative indications of stimulated macropinocytosis, a lipid-raft-dependent and receptor-independent form of fluid-phase endocytosis [24], have been implicated for the CPPs Tat and oligoarginines [25,26]. This study will build on the classic 16 residue basic and amphipathic CPP penetratin, derived from the Antennapedia homeodomain in Drosophila [27,28], and on two analogs, PenArg and PenLys (see Table 1 for peptide sequences), designed by us to systematically explore the impact of arginines and lysines on CPP characteristics [13,23,29,30].
Materials and methods Materials. Fluorescein isothiocyanate (FITC) labelled peptides (>80% purity) were purchased from NeoMPS (San Diego). The dye was conjugated to the N-terminus via a gamma-amino butyric acid (GABA) linker. Texas red-labelled 70 kDa neutral dextran (TR-dextran) and FM 4-64 were from Invitrogen, and cell culture reagents from PAA Laboratories. Chinese hamster ovarian cells (CHO-K1) were a kind gift from Prof. Ülo Langel, Stockholm University. Cell culture. CHO-K1 cells were cultured in HAM’s F12 medium supplemented with fetal calf serum (10%) and L-glutamine (2 mM) at 37 °C and 5% CO2. The cells were trypsinized every 3–4 days and seeded at a density of 7500 cells/cm2. For quantitative uptake experiments cells were seeded in 12-well plates
622
H.L. Åmand et al. / Biochemical and Biophysical Research Communications 371 (2008) 621–625
Table 1 Peptide sequences Peptide
Sequence
Penetratin PenArg PenLys
FITC-GABA-RQIK IWFQ NRRM KWKK-NH2 FITC-GABA-RQIR IWFQ NRRM RWRR-NH2 FITC-GABA-KQIK IWFQ NKKM KWKK-NH2
Arginines are marked in bold and lysines are underlined.
(11000 cells/cm2) and cultured to 90–100% confluence. For confocal imaging, cells were seeded on round coverslips at a density of 11000 cells/cm2 and cultured for 2 days. Quantitative uptake. Cells were rinsed with serum-free HAM’s F12 and incubated with appropriate reagents in serum-free HAM’s F12 for 1 h (1, 5 or 10 lM peptide and/or 100 lg/ml TR-dextran or 5 lg/ml FM 4-64). The cells were rinsed twice with Hepes buffered saline (20 mM HEPES, 150 mM NaCl, pH 7.4) supplemented with 100 lg/ml heparin (10 times the concentration required to block uptake of the similarly charged CPP Tat [26]) to remove externally bound peptide. The cells were collected by trypsination (10 min) followed by centrifugation (5 min, 5000g, 4 °C) and thereafter lysed (0.1 M NaOH, on ice, 1 h). Cell lysates were transferred to black 96-well plates for fluorimetric quantification of internalized CPP and endocytosis marker. A FluoStar Optima Plate Reader (BMG Labtechnologies, Germany) with excitation/emission filter settings at 485/520 nm for FITC-labelled peptides and at 544/590 nm for TR-dextran and FM 4-64 was used. The total protein concentration in each sample was determined using a protein assay kit from BioRad. The protocol essentially follows reference [31]. All experiments were repeated at least three times and always performed in triplicate. The uptake levels are reported as the arithmetic mean of all samples, whereas the error bars represent the average maximum and minimum data spread. All data were corrected for background contributions by subtracting appropriate blanks and normalized against the total protein concentration in each sample. Confocal imaging. Cells for confocal imaging were rinsed with serum-free HAM’s F12 and incubated for 1 h at 37 °C with appropriate reagents. Prior to imaging cells were washed again and the coverslips were mounted with fresh media in a solution chamber. A confocal laser scanning microscopy system (Leica TCS SP2 RS, Wetzlar, Germany), with a PL APO 63/1.32 objective, was used for acquisition of confocal fluorescence images. The 488 nm line of an argon laser was used for excitation of FITC and the 543 and 594 nm lines of a helium–neon laser were used for excitation of FM 4-64 and TR-dextran, respectively. Images were acquired in sequential mode to avoid bleed-through between the photomultiplier tubes. Detector settings (photomultiplier high tension) and contrast were optimized for each sample.
Results Quantification of CPP uptake Fig. 1 shows the uptake of FITC-labelled PenLys, penetratin, and PenArg in live CHO-K1 cells obtained after 1 h incubation at 37 °C. Uptake rate is in the order PenArg > penetratin > PenLys, indicating
Fig. 1. Uptake of penetratin peptides after 1 h incubation at 37 °C as a function of peptide concentration. Column height represents the average of nine independent experiments, performed in triplicate (N = 9, n = 3). Error bars indicate the average maximum and minimum deviation in each experiment (see text for further details).
that arginine-rich peptides are more efficiently internalized than lysine-rich ones. PenLys is however internalized in CHO-K1 cells (see also Fig. 2A), a finding that disputes previous observations and will be further considered in the Discussion. The uptake efficiency is roughly linearly dependent on peptide concentration and uptake is not saturable within this concentration regime. Care was taken to avoid measuring ‘‘false” fluorescence from externally bound peptide: cells were rinsed twice with HBS buffer supplemented with excess amounts of negatively charged heparin (see Materials and methods). Thereafter, cells were subjected to trypsination, a procedure that should remove any residual bound peptide and also help in degrading heparin-sequestered peptide, preventing resorption to the cell surface during the centrifugation step. Confocal imaging To confirm the intracellular location of peptide (vesicular versus cytoplasmic), confocal laser images were obtained on CHO-K1 cells incubated with CPPs and/or endocytosis markers for 1 h (Fig. 2). Fig. 2A shows peptide uptake and co-localization with the nonspecific marker of endocytosis FM 4-64. The detector settings and contrast were adjusted for best visualization of each image and the intensities are therefore not comparable. Intracellular fluorescence is observed for all three peptides and the punctuate pattern strongly indicates vesicular localization within the cell, thus confirming endocytotic uptake. Substantial, but not perfect, colocalization with FM 4-64 is observed (orange/yellow color), indicating that not all endocytotic vesicles contain peptide. Fig. 2B shows uptake of TR-dextran, a fluid-phase endocytosis marker used to indicate macropinocytosis, in absence and presence of peptide. TR-dextran is negligibly internalized in absence of peptide, whereas even the least well-internalized peptide (PenLys) markedly increases its uptake. TR-dextran co-localizes well with peptide, but some vesicular structures containing only peptide are also observed. Fig. 2C shows uptake of PenArg at an elevated concentration (10 lM). Both diffuse cytoplasmic and punctuate fluorescence are observed, but the transmitted image indicates that these cells have severely compromised morphology. This micrograph was included to demonstrate that PenArg is toxic at elevated concentrations. Such behavior was not observed for penetratin or PenLys under any conditions (data not shown). Quantification of stimulated endocytosis To assess to what extent penetratin peptides can stimulate endocytosis, quantitative measurements were performed where peptide was co-incubated with FM 4-64 or TR-dextran. FM 4-64 is a non-specific endocytosis marker that fluoresces only when bound to membranes. It cannot translocate across the plasma membrane and is only internalized via endocytosis. TR-dextran is used as marker of fluid-phase endocytosis. Dextrans of this size (70 kDa) are preferentially internalized by macropinocytosis compared to micropinocytotic mechanisms including clathrin-, caveolae-, and receptor-mediated endocytosis [32]. Fig. 3 shows intrinsic cell uptake of FM 4-64 (control) and uptake in presence of peptide. No statistically significant increase in uptake of FM 4-64 can be detected for PenLys or for penetratin and PenArg applied at 1 lM concentration. Some increased uptake is observed for the highest penetratin concentration and for PenArg applied at 5 and 10 lM. For PenArg, 10 lM, we cannot exclude that the exaggerated internalization is not due to peptide-mediated permeabilization of the plasma membrane (see Fig. 2C). Fig. 4 shows uptake of TR-dextran in presence of peptide. TR-dextran is not efficiently internalized in CHO-K1 cells under normal conditions (see also Fig. 2B). In presence of peptide, uptake is markedly increased, even at 1 lM peptide concentration. The amount of internalized TR-dextran
H.L. Åmand et al. / Biochemical and Biophysical Research Communications 371 (2008) 621–625
623
Fig. 2. Confocal images of live CHO-K1 cells incubated with peptide and/or endocytosis markers. (A) Uptake and co-localization (yellow indicates very good overlap) of FITClabelled peptide (green) and non-specific endocytosis marker FM 4-64 (red). The peptide concentration was 5 lM and the FM 4-64 concentration was 5 lg/ml. (B) Uptake of TR-dextran (50 lg/ml) in absence and presence of peptide (5 lM PenLys). (C) Uptake and distribution of PenArg applied at 10 lM concentration. Note the compromised cell morphology. Images were acquired after 1 h incubation at 37 °C. The image contrast and the gain (high tension over the photomultiplier tube detectors) have been optimized for each micrograph and the intensities in each image are thus not comparable.
Fig. 3. Internalization of the non-specific endocytosis marker FM 4-64 in absence (control) and presence of penetratin peptides after 1 h incubation at 37 °C. The FM 4-64 concentration was 5 lg/ml. Column heights represents the average of three independent experiments, performed in triplicate (N = 3, n = 3). Error bars represent the average maximum and minimum deviation in each experiment (see text for details).
Fig. 4. Internalization of fluid-phase endocytosis marker TR-dextran in absence (control) and presence of penetratin peptides. The cells were incubated for 1 h at 37 °C. The TR-dextran concentration was 100 lg/ml. Column heights represents the average of three independent experiments, performed in triplicate (N = 3, n = 3). Error bars represent the average maximum and minimum deviation in each experiment (see text for details).
Discussion increases with peptide concentration and is more efficient for PenArg than for penetratin and PenLys. Control experiments verified that neither TR-dextran nor FM 4-64 affected the degree of peptide internalization (data not shown).
Despite the fact that almost 20 years have elapsed since the idea of cell-penetrating peptides as transporters emerged, our understanding of how cell-penetrating peptides like penetratin, Tat, or oligoarginine, enter cells is still elusive from several aspects (see
624
H.L. Åmand et al. / Biochemical and Biophysical Research Communications 371 (2008) 621–625
Introduction). Here we shed some light on the CPP uptake mechanisms by demonstrating how penetratin triggers its own internalization by stimulating endocytosis and by quantifying the relative importance of arginines versus lysines for the efficiency of this process. Arginine-rich peptides have been argued to display superior cell-penetrating abilities compared to their lysine-rich counterparts, some reports even indicate a difference so extreme that whilst arginine-rich CPPs enter cells efficiently, the corresponding lysine-rich versions are completely unable to do so [13,17]. This study proves that both lysine-rich (PenLys) and arginine-rich (PenArg) versions of penetratin can enter CHO-K1 cells, albeit the former less efficiently (Fig. 1). This study is in conflict with some previous results, particularly those of a live cell confocal imaging study on PenLys [13], but our results are consistent with biophysical studies of peptide–membrane interaction in cell membrane mimetic systems [6,23,29] and also understandable from a physico-chemical perspective (vide infra). We have confirmed that PenLys cell-penetrating ability is not restricted to CHO-K1, but also occurs in at least two other cell lines: NIH-3T3 mouse fibroblasts and NG108-15 neuroblastoma/rat glioma cells (unpublished observations). The uptake efficiency of PenArg exceeds that of PenLys with about one order of magnitude. This difference can partly be explained by PenArg’s greater affinity for lipid membranes, but reported surface partition coefficients (PenArg 1900 M 1 and PenLys 600 M 1 for binding to lipid vesicles with 40% negative charge [30]) are not sufficiently different to entirely explain a 10fold difference in internalization. One may argue, based on previous suggestions, that negatively charged heparan sulfates are the primary cell surface binding sites for CPPs [33–35], that a lipid bilayer is not a suitable model for CPP cell interaction. However, since the negatively charged entity in HS is an oxoanion (SO4 ) just as the lipid phosphate (PO4 ) and since penetratin binds to lipid bilayers primarily by electrostatic interactions, we think it can be justified to assume that binding affinity for HS will fall in the same order for the penetratins. This is certainly the case for at least one other negatively charged polyelectrolyte, namely DNA (Åmand et al., in progress). Therefore, whilst CPP cell surface affinity in part explains uptake efficiency we believe that also other factors, such as ability to trigger cellular response, must be involved in order to fully explain the different uptake characteristics of penetratin, PenArg, and PenLys. Which these factors could be and how they relate to the chemistry of arginines and lysines remain to be explored. The peptide internalization observed in the quantitative uptake assay (Fig. 1) was verified to be mainly endocytotic (Fig. 2A). Not even PenArg, which has previously been indicated to use direct membrane penetration mechanisms [13], displays any significant diffuse cytoplasmic fluorescence except at the highest applied concentration (10 lM) (Fig. 2). Two previous reports have indicated that the CPPs Tat and octaarginine stimulate macropinocytosis [26,36] and Khalil et al. estimate roughly a 40% increase in macropinocytotic activity for their octaarginine–liposome constructs [36]. This study proves that also penetratin peptides trigger their own internalization by stimulating fluid-phase uptake of a large molecular weight marker (TR-dextran) used as indicator for macropinocytosis. Taking a step further, this study also quantifies to what extent this mechanism is up-regulated and shows that all three peptides increase uptake of TR-dextran significantly (up to ten times). PenLys, 1 lM, is the least effective agent but still doubles the uptake. The efficiency comes, as for peptide internalization, in the order PenArg > penetratin > PenLys suggesting that these two events are directly related. The co-localization of peptide and TR-dextran is however not perfect (Fig. 2B); some internalized vesicular structures contain only peptide, but the opposite is much
more infrequent, showing in accordance with previous reports [1] that penetratin is not exclusively internalized by one type of endocytosis. This raises an imperative question which we will attempt to answer: Does penetratin stimulate only its own uptake, i.e. will endocytosis occur only at sites where the peptide binds, or does cell surface interactions initiate signalling cascades that increase the endocytotic activity of the entire cell? Fig. 3 gives some information to this end, showing that the penetratin peptides have only marginal effects on the overall endocytosis rate (measured using a non-specific endocytosis marker) compared to the effect on the macropinocytotic activity. For 10 lM penetratin and 5 lM PenArg the amount of internalized FM 4-64 has barely doubled (compared to an 8–10 times increased TR-dextran uptake under similar conditions). At even lower penetratin or PenArg concentrations and for PenLys the change in endocytotic activity is negligible. Thus, we conclude that penetratin peptides predominantly stimulate their own uptake by initiating macropinocytotic fluid-phase uptake in CHO-K1. Macropinocytosis initiates with cell membrane ruffling and thus it is reasonable to conclude that penetratin stimulates this activity by its cell surface interactions. It appears as if up-regulation of macropinocytosis to some extent occurs at the expense of other endocytotic pathways since uptake of FM 4-64 is only marginally affected. Possibly the cell strives at keeping its membrane turnover rate relatively constant. In conclusion, this study evidences that penetratin peptides can trigger their own uptake by stimulating macropinocytosis upon binding to the cell surface of CHO-K1 cells. We also find indications for that up-regulation of this pathway occurs on the expense of other endocytotic mechanisms. Further, comparison of PenArg, penetratin and PenLys confirms that incorporation of additional arginines into a CPP increases its efficiency, but the results obtained for PenArg raise the question whether ‘‘better” will also mean ‘‘more toxic”. Support for an increased toxicity of argininerich CPPs, compared to penetratin, can be found in one of few comparative CPP toxicity studies [37] and possibly penetratin owes its efficient and non-toxic CPP properties to a balanced mix of arginines and lysines. Acknowledgments This work was supported by funding from the Swedish Cancer Society and from the European Commission (contracts 012967, 005204 and 037783). Prof. Langel at Department of Neurochemistry, Stockholm University, Sweden is acknowledged for the kind gift of the CHO-K1 cells. References [1] Ulo Langel Handbook of Cell-Penetrating Peptides, second ed., CRC Press, Taylor & Francis Group, Boca Raton, London, New York, 2007. [2] P.E. Thorén, D. Persson, M. Karlsson, B. Nordén, The antennapedia peptide penetratin translocates across lipid bilayers—the first direct observation, FEBS Lett. 482 (2000) 265–268. [3] E. Barany-Wallje, S. Keller, S. Serowy, S. Geibel, P. Pohl, M. Bienert, M. Dathe, A critical reassessment of penetratin translocation across lipid membranes, Biophys. J. 89 (2005) 2513–2521. [4] J. Björklund, H. Biverståhl, A. Gräslund, L. Mäler, P. Brzezinski, Real-time transmembrane translocation of penetratin driven by light-generated proton pumping, Biophys. J. 91 (2006) L29–L31. [5] M. Magzoub, A. Pramanik, A. Gräslund, Modeling the endosomal escape of cellpenetrating peptides: transmembrane pH gradient driven translocation across phospholipid bilayers, Biochemistry 44 (2005) 14890–14897. [6] D. Persson, P.E. Thorén, E.K. Esbjörner, M. Goksör, P. Lincoln, B. Nordén, Vesicle size-dependent translocation of penetratin analogs across lipid membranes, Biochim. Biophys. Acta 1665 (2004) 142–155. [7] D. Terrone, S.L. Sang, L. Roudaia, J.R. Silvius, Penetratin and related cellpenetrating cationic peptides can translocate across lipid bilayers in the presence of a transbilayer potential, Biochemistry 42 (2003) 13787–13799. [8] P.E. Thorén, D. Persson, E.K. Esbjörner, M. Goksör, P. Lincoln, B. Nordén, Membrane binding and translocation of cell-penetrating peptides, Biochemistry 43 (2004) 3471–3489.
H.L. Åmand et al. / Biochemical and Biophysical Research Communications 371 (2008) 621–625 [9] M. Lundberg, M. Johansson, Positively charged DNA-binding proteins cause apparent cell membrane translocation, Biochem. Biophys. Res. Commun. 291 (2002) 367–371. [10] J.P. Richard, K. Melikov, E. Vives, C. Ramos, B. Verbeure, M.J. Gait, L.V. Chernomordik, B. Lebleu, Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake, J. Biol. Chem. 278 (2003) 585–590. [11] A. Fischer, T. Oberholzer, P.L. Luisi, Giant vesicles as models to study the interactions between membranes and proteins, Biochim. Biophys. Acta 1467 (2000) 177–188. [12] D.J. Mitchell, D.T. Kim, L. Steinman, C.G. Fathman, J.B. Rothbard, Polyarginine enters cells more efficiently than other polycationic homopolymers, J. Pept. Res. 56 (2000) 318–325. [13] P.E. Thorén, D. Persson, P. Isakson, M. Goksör, A. Önfelt, B. Nordén, Uptake of analogs of penetratin, Tat(48–60) and oligoarginine in live cells, Biochem. Biophys. Res. Commun. 307 (2003) 100–107. [14] F. Duchardt, M. Fotin-Mleczek, H. Schwarz, R. Fischer, R. Brock, A comprehensive model for the cellular uptake of cationic cell-penetrating peptides, Traffic 8 (2007) 848–866. [15] M.M. Fretz, N.A. Penning, S. Al-Taei, S. Futaki, T. Takeuchi, I. Nakase, G. Storm, A.T. Jones, Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells, Biochem. J. 403 (2007) 335–342. [16] M. Fotin-Mleczek, R. Fischer, R. Brock, Endocytosis and cationic cellpenetrating peptides–a merger of concepts and methods, Curr. Pharm. Des. 11 (2005) 3613–3628. [17] J.B. Rothbard, T.C. Jessop, R.S. Lewis, B.A. Murray, P.A. Wender, Role of membrane potential and hydrogen bonding in the mechanism of translocation of guanidinium-rich peptides into cells, J. Am. Chem. Soc. 126 (2004) 9506– 9507. [18] M. Nishihara, F. Perret, T. Takeuchi, S. Futaki, A.N. Lazar, A.W. Coleman, N. Sakai, S. Matile, Arginine magic with new counterions up the sleeve, Org. Biomol. Chem. 3 (2005) 1659–1669. [19] S. Futaki, T. Suzuki, W. Ohashi, T. Yagami, S. Tanaka, K. Ueda, Y. Sugiura, Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery, J. Biol. Chem. 276 (2001) 5836–5840. [20] N. Sakai, S. Matile, Anion-mediated transfer of polyarginine across liquid and bilayer membranes, J. Am. Chem. Soc. 125 (2003) 14348–14356. [21] N. Sakai, T. Takeuchi, S. Futaki, S. Matile, Direct observation of anionmediated translocation of fluorescent oligoarginine carriers into and across bulk liquid and anionic bilayer membranes, Chembiochem. 6 (2005) 114–122. [22] J.B. Rothbard, T.C. Jessop, P.A. Wender, Adaptive translocation: the role of hydrogen bonding and membrane potential in the uptake of
[23]
[24] [25]
[26]
[27]
[28]
[29]
[30] [31] [32]
[33]
[34] [35] [36]
[37]
625
guanidinium-rich transporters into cells, Adv. Drug. Deliv. Rev. 57 (2005) 495–504. E.K. Esbjörner, P. Lincoln, B. Nordén, Counterion-mediated membrane penetration: cationic cell-penetrating peptides overcome Born energy barrier by ion-pairing with phospholipids, Biochim. Biophys. Acta 1768 (2007) 1550– 1558. S.D. Conner, S.L. Schmid, Regulated portals of entry into the cell, Nature 422 (2003) 37–44. A. Iwasa, H. Akita, I. Khalil, K. Kogure, S. Futaki, H. Harashima, Cellular uptake and subsequent intracellular trafficking of R8-liposomes introduced at low temperature, Biochim. Biophys. Acta 1758 (2006) 713–720. J.S. Wadia, R.V. Stan, S.F. Dowdy, Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis, Nat. Med. 10 (2004) 310–315. A. Joliot, C. Pernelle, H. Deagostini-Bazin, A. Prochiantz, Antennapedia homeobox peptide regulates neural morphogenesis, Proc. Natl. Acad. Sci. USA 88 (1991) 1864–1868. D. Derossi, A.H. Joliot, G. Chassaing, A. Prochiantz, The third helix of the Antennapedia homeodomain translocates through biological membranes, J. Biol. Chem. 269 (1994) 10444–10450. C.E. Caesar, E.K. Esbjörner, P. Lincoln, B. Nordén, Membrane interactions of cell-penetrating peptides probed by tryptophan fluorescence and dichroism techniques: correlations of structure to cellular uptake, Biochemistry 45 (2006) 7682–7692. D. Persson, P.E.G. Thorén, P. Lincoln, B. Nordén, Vesicle membrane interactions of penetratin analogues, Biochemistry 43 (2004) 11045–11055. T. Holm, H. Johansson, P. Lundberg, M. Pooga, M. Lindgren, Ü. Langel, Studying the uptake of cell-penetrating peptides, Nat. Protoc. 1 (2006) 1001–1005. N. Araki, M.T. Johnson, J.A. Swanson, A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages, J. Cell Biol. 135 (1996) 1249–1260. M. Tyagi, M. Rusnati, M. Presta, M. Giacca, Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans, J. Biol. Chem. 276 (2001) 3254– 3261. S.M. Fuchs, R.T. Raines, Pathway for polyarginine entry into mammalian cells, Biochemistry 43 (2004) 2438–2444. M. Belting, Heparan sulfate proteoglycan as a plasma membrane carrier, Trends Biochem. Sci. 28 (2003) 145–151. I.A. Khalil, K. Kogure, S. Futaki, H. Harashima, High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression, J. Biol. Chem. 281 (2006) 3544–3551. S.W. Jones, R. Christison, K. Bundell, C.J. Voyce, S.M. Brockbank, P. Newham, M.A. Lindsay, Characterisation of cell-penetrating peptide-mediated peptide delivery, Br. J. Pharmacol. 145 (2005) 1093–1102.