Membrane leakage induced by dynorphins

FEBS Letters 580 (2006) 3201–3205

Membrane leakage induced by dynorphins Loı¨c Hugonina, Vladana Vukojevic´b, Georgy Bakalkinb, Astrid Gra¨slunda,* b

a Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, S-10691 Stockholm, Sweden Section of Alcohol and Drug Dependence Research, Department of Clinical Neuroscience, Karolinska Institute, S-17176 Stockholm, Sweden

Received 6 December 2005; revised 24 April 2006; accepted 25 April 2006 Available online 4 May 2006 Edited by Maurice Montal

The endogenous dynorphin peptides including dynorphin A 1–17 (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn), which consists of Dyn A and Dyn B sequences, are ligands for j-opioid receptors. They are synthesized in the striatum, amygdala, hippocampus and other brain structures and also in the spinal cord [1–8]. Consistent with their anatomical localization, these peptides play a role in the modulation of reward induced by intake of addictive substances, motor control, stress response, memory acquisition and pain processing [9–12]. Several actions of Dyn A and Big Dyn have been found to be insensitive to the general opioid antagonist naloxone and the selective j-antagonist nor-binaltorphimine [12–15]. The non-opioid excitotoxic effects of the synthetic and endogenous Dyn A and Big Dyn can result in neurodegeneration and neuronal death and are relevant for pathophysiological processes such as chronic neuropathic pain and spinal cord and brain injury [13,16–20]. Non-opioid dynorphin actions can be blocked

by antagonists of the N-methyl-D -aspartate (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate (AMPA) glutamate receptors [12–16,19–21]. The glutamate receptors are involved but do not apparently represent the primary targets for Dyn A and Big Dyn. Cell imaging experiments demonstrated that Dyn A can induce a time- and dose-dependent increase in intra-neuronal calcium concentration through a non-opioid [22], non-glutamate receptor mediated mechanism [23]. This mechanism may underlie nonopioid neuropathological effects of dynorphins. We recently demonstrated that dynorphins share common properties with cell penetrating peptides (CPPs) [24]. CPPs are short, 5–35 amino acid peptides capable to translocate across the cell plasma membrane without a chiral receptor [25]. Recent studies indicate that CPPs may be transported into cells through macropinocytosis, a form of endocytosis [26]. Macropinocytosis may operate in parallel with the direct translocation of these peptides across the plasma membrane [27,28]. Several mechanisms have been proposed to explain direct CPP translocation including pore formation [29], carpet [29,30], membrane-thinning [31] or inverted micelles models [32,33]. Similarly to CPPs, Dyn A and Big Dyn can translocate across plasma membranes of live neurons and non-neuronal cells [24]. Big Dyn has a much higher potency to penetrate than Dyn A, which is similar in this respect to a prototypical CPP transportan10. Dyn B and the central 22-amino acid Big Dyn fragment did not penetrate into cells [24]. Translocation of dynorphins into cells followed by their interactions with intracellular targets was suggested as an evolutionary ancient mechanism of intercellular communication and signal transduction relevant for the non-opioid actions of these peptides. The mechanisms of dynorphin transport across the plasma membrane are unknown. One possibility is that dynorphins can make transient pores in the lipid bilayer or induce membrane perturbations allowing them to translocate into cells. To test this hypothesis we investigated the interactions of dynorphins with phospholipid membranes in the present study. Peptide interactions with large unilamellar phospholipid vesicles (LUVs) of varying surface charge densities were evaluated using fluorescence spectroscopic methods. The membrane perturbation effects of the peptides were assessed by calcein leakage experiments.

* Corresponding author. Fax: +46 8 155597. E-mail address: [email protected] (A. Gra¨slund).

2. Materials and methods

Abbreviations: CPP, cell penetrating peptide; Big Dyn, big dynorphin; Dyn A, Dynorphin A; Dyn B, Dynorphin B; LUVs, large unilamellar phospholipid vesicles; POPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine; POPG, 1-palmitoyl-2-oleoyl-phosphatidylglycerol

2.1. Materials Dyn A was produced by Neosystem Laboratoire, Strasbourg. The identity and purity was controlled by amino acid, mass spectral and HPLC analysis. Big Dyn, and Dyn B (Table 1) were synthesized at

Abstract Dynorphins, endogeneous opioid peptides, function as ligands to the opioid kappa receptors and induce non-opioid excitotoxic effects. Here we show that big dynorphin and dynorphin A, but not dynorphin B, cause leakage effects in large unilamellar phospholipid vesicles (LUVs). The effects parallel the previously studied potency of dynorphins to translocate through biological membranes. Calcein leakage caused by dynorphin A from LUVs with varying POPG/POPC molar ratios was promoted by higher phospholipid headgroup charges, suggesting that electrostatic interactions are important for the effects. A possibility that dynorphins generate non-opioid excitatory effects by inducing perturbations in the lipid bilayer of the plasma membrane is discussed. Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Dynorphin; Cell penetrating peptides; Phospholipid membranes; Unilamellar vesicles; Fluorescence; Calcein leakage

1. Introduction

0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.04.078

3202

L. Hugonin et al. / FEBS Letters 580 (2006) 3201–3205

Table 1 Amino acid sequences of the studied peptides Peptide

Sequence

Charge at pH 7.0

Dyn A Dyn B Big Dyn

YGGFLRRIRPKLKWDNQ YGGFLRRQFKVVT YGGFLRRIRPKLKWDNQKRYGGFLRRQFKVVT

+4 +3 +9

The peptides have native N- and C-termini.

2.3. Preparation of large unilamellar vesicles LUVs were prepared by initially dissolving the phospholipids at the desired concentration (with the chosen POPG/POPC molar ratio) in a chloroform/ethanol mixture, to ensure the complete mixing of the components, and then removing the solvent by placing the sample under N2 (g) until the lipids were completely dried. The dried lipids were dispersed in 50 mM potassium phosphate buffer (pH 7.2). The dispersion was run through a freeze–thaw cycle five times and then passed through two polycarbonate filters (0.1 lm pore size) 20 times in an Avanti manual extruder. 2.4. Calcein leakage LUVs with entrapped calcein were prepared by hydrating a dried lipid film of the desired composition with 70 mM calcein present in the buffer (final pH was adjusted to 7.2 by addition of NaOH from a 10 M stock solution). Calcein not entrapped was separated from the LUVs on a Sephadex-G25 column. Fluorescence was measured on a Horiba Jobin Yvon Fluorolog-3 spectrometer using the DataMax operating software. The fluorescence from calcein at 70 mM concentration was low due to self-quenching, but increased considerably upon dilution. Increasing concentrations of peptides were added to LUVs composed of 100 lM phospholipid mixtures of defined POPG/POPC content. After 10 min incubation at 25 °C, the release of calcein from the LUVs was monitored by an increase in the fluorescence intensity. The fluorescence intensity was measured at 520 nm (excitation at 490 nm). The maximum fluorescence intensity (100% leakage) was determined by lysing the vesicles with a 10% (w/v) solution of the detergent Triton X-100. The percent leakage value was then calculated according to the following equation:   F  F0 % ð1Þ percent leakage ¼ 100 F max  F 0 where F0 represents the fluorescence intensity of the intact vesicle, F and Fmax the intensity before and after the addition of the detergent, respectively. The experiments were generally repeated at least three times with different preparations of vesicles. The experimental uncertainty was estimated to ±5% of the measured value.

3.1. Leakage of Calcein from LUVs The membrane perturbation effects of the peptides were investigated through LUV calcein leakage experiments, evaluated using Eq. 1. The peptides studied here had native N- and C-termini. In the absence of the peptide, no leakage of calcein from the LUVs was observed. Fig. 1A shows the % leakage induced by the peptides from LUVs with partially negatively charged headgroups, POPG/POPC (30/70), after 10 min incubation at 25 °C. Both Big Dyn and Dyn A cause a substantial degree of leakage and for both peptides the leakage is almost satured at P/L = 0.05 corresponding to 5 lM peptide concen-

A

100 80

% Leakage

2.2. Determination of peptide concentrations The concentration of peptides in aqueous stock solutions was determined spectrophotometrically at k = 280 nm on a CARY 4 spectrophotometer. A molar absorptivity at 280 nm of 5690 M1 cm1 for one Trp and 1280 M1 cm1 for one Tyr residue was applied in the calculations [34].

3. Results

60 40 20 0 0

0.2

0.4

P/L B 100 80

% Leakage

the Department of Medical Biochemistry and Microbiology, Uppsala University. The peptides were not modified in the termini. The peptides were purified by reversed-phase chromatography on Vydac C18 218 TP 1022 and Sephasil C8 columns in a 0.1% trifluoroacetic acid acetonitrile/water solvent system and finally by gel filtration on a Superdex column in 1 M acetic acid. They were analyzed by analytical reversedphase chromatography and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Purity was determined by integration of the UV peak at 215 nm. The purity of all peptides was >98%. Melittin from the venom of honeybee (Apis mellifera) was obtained from Sigma. 1-Palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and 1-palmitoyl-2-oleoyl-phosphatidyl-glycerol (POPG) were purchased from Avanti Polar Lipids, Alabaster, of the best quality, and used without further purification. Calcein, a fluorescein derivative (C30H26N2O13, Mw = 622.5 g/mol), was purchased from Molecular Probes, The Netherlands.

60 40 20 0 0

2

4

6

8

10

Time (min) Fig. 1. (A) Calcein leakage from LUVs, induced by the peptides: Dyn A (s), Big Dyn (d), Dyn B (j) and melittin (h). Increasing concentrations of the peptides were added to LUV samples, composed of 100 lM phospholipids, mixed as POPG/POPC [30/70]. The LUVs had 70 mM calcein entrapped before adding the peptide. The % calcein release after 10 min was plotted as a function of total peptide-to-lipid molar ratio, P/L. The solvent was 50 mM phosphate buffer (pH 7.2). The calcein fluorescence was recorded at an ambient temperature (25 °C). (B) Time-dependence of calcein leakage due to addition of peptides at P/L = 0.05, with other conditions as in A.

L. Hugonin et al. / FEBS Letters 580 (2006) 3201–3205

3203

tration. Big Dyn induces a higher degree of leakage than Dyn A (70% and 40%, respectively). Dyn B induced no leakage. The pore-forming peptide melittin was used as a comparison, and was found to induce almost 100% leakage already at a P/ L  0.02. The time dependence study of calcein leakage from the vesicles at a P/L ratio of 0.05 (Fig. 1B) indicated that after 10 min almost no further release of calcein was observed. Consequently the percentage of leakage after 10 min presented in Fig. 1 should approximately represent the final stage at each peptide concentration. In another series of experiments the influence of LUV headgroup charges was studied for Dyn A. The leakage caused by Dyn A interaction from LUVs of different POPG/POPC molar ratio compositions is presented in Fig. 2. The peptide causes leakage (40%) in the zwitterionic membrane POPC LUVs, similar to the charged LUVs with POPG/POPC (30/70). Between P/L ratios 0 and 0.1 the leakage is less prominent with the zwitterionic LUVs. In the more highly charged LUVs with POPG/POPC (50/50), Dyn A causes a higher degree of leakage (80%). The data suggest a sigmoidal dependency of leakage intensity versus Dyn A concentration for the zwitterionic POPC LUVs (Fig. 2), possibly related to membrane-mediated cooperative interactions leading to pore formation [35]. The dependence on phospholipid headgroup charge shows that electrostatic interactions affect the processes. When the surface charge on the membrane is increased, the peptide–membrane interactions are apparently increased, and the leakage profile becomes hyperbolic. Previous studies have shown that the dynorphins interact strongly with small unilamellar phospholipid vesicles (SUVs), however with only a surprisingly weak induction of secondary structure [24]. We have here observed similar weak structure induction by CD spectroscopy for the dynorphins using the LUV membrane model systems (data not shown). This is in contrast to most amphipathic peptides [36], and indeed a simple helical wheel study shows very little amphipathic character of the dynorphin sequences (data not shown).

100

% Leakage

80 60 40 20 0 0

0.2

0.4

P/L Fig. 2. Dyn A-induced leakage from LUVs, composed of 250 lM phospholipids of different POPG/POPC molar ratios: POPG/POPC [0/ 100] (s), POPG/POPC [30/70] (h), POPG/POPC [50/50] (d).The LUVs had 70 mM calcein entrapped before adding the peptide. The % calcein release after 10 min incubation was plotted as a function of the total peptide-to-lipid molar ratio, P/L. The solvent was 50 mM phosphate buffer (pH 7.2). The calcein fluorescence was recorded at an ambient temperature (25 °C).

4. Discussion The dynorphins are similar to several groups of CPPs in the high content of hydrophobic and basic amino acids (Table 1). The generally low cytotoxic effects of some CPPs, such as penetratin peptide (pAntp), distinguish them from translocating cationic antimicrobial and lytic peptides, such as melittin, which have strong effects on the membrane integrity causing leakage effects and have a powerful hemolytic activity [37]. However, certain other CPPs, such as the model amphipathic peptide (MAP) and transportan, exhibit membrane toxicity in cells [38]. Our previous studies have established that Big Dyn and to a lesser extent Dyn A, but not Dyn B, efficiently translocate through cell membranes of neuronal and nonneuronal cells [24]. Big Dyn and Dyn A, but not Dyn B, also exhibit cytotoxicity in non-neuronal and neuronal cells [39]. The present study of the dynorphins in membrane model systems was undertaken to elucidate the molecular mechanisms by which the dynorphins exert their non-opioid activities. The results show that Big Dyn and Dyn A, but not Dyn B, cause perturbations and leakage in a membrane model system composed of phospholipid LUVs. The higher membrane-perturbing potency of Big Dyn compared to Dyn A may be due to its higher positive charge (Table 1). The low potency of Dyn B may be due to its smaller positive charge, or possibly its shorter length compared to the other dynorphins. The fact that Dyn B has the highest percentage of hydrophobic residues of the three peptides apparently does not make it more potent as a membrane-perturbing agent. The calcein leakage from LUVs could be induced either by making channels in them (pore-formation) or by lysis of the vesicles. The latter is unlikely since the vesicles exhibit only partial leakage in presence of high concentrations of either Big Dyn or Dyn A. Several other studies on peptides interacting with membranes have reported similar saturation effects, yielding leakage curves which seem to level off as a function of time at constant peptide concentration [40,41]. This phenomenon has been explained in terms of transient pore formation with a nucleation step during an initial bilayer perturbation period, followed by a transient restabilization of the peptide/lipid bilayer structure, which gives rise to the observed leveling off of the spontaneous leakage [42]. The kinetics of leakage of Big Dyn and Dyn A can be explained in similar terms. Melittin is known to make transient or more permanent pores in phospholipid membranes [43,44]. Big Dyn and Dyn A may cause similar effects although they are less potent in their membrane perturbation. The most important observation in the present study is that the potency to translocate through cell membranes [24], the cytotoxicity [37] and the degree of leakage from LUVs induced by the dynorphins, all follow the same order and seem to be correlated when comparing the three peptides. Consistent with these in vitro data, Big Dyn administered intrathecally into mice induces characteristic nociceptive behavior insensitive to naloxone, whereas Dyn A was 100-fold less potent and Dyn B was inactive [19]. These results suggest a close molecular connection between the phenomena, which hypothetically involves the formation of dynorphin-induced transient membrane pores, similar to those induced by melittin. The high positive charge has been demonstrated to be important for the binding of CPPs to negatively charged gly-

3204

cosaminoglycans on the plasma membrane [26]. This may be important also for the dynorphin activities. The study of Dyn A interaction with LUVs of different POPG/POPC compositions suggest that the membrane interaction is not only due to charge interactions, since the peptide causes leakage also in vesicles with zwitterionic headgroups. However, higher membrane charge density results in higher degree of leakage as has been observed also for other positively charged peptides [41]. Consequently electrostatic but also hydrophobic interactions are important for the binding of Dyn A to membranes. In conclusion, the mechanism of membrane leakage induced by dynorphins is not yet understood on the molecular level but there are indications that formation of pores is important. The correlation between results obtained with phospholipid membranes, on live cells in vitro [24,39] and in animal experiments [19,20] suggests that dynorphins induce non-opioid excitatory effects through transient pore formation or perturbations of the phospholipid bilayer in the plasma membrane. These pores or perturbations may allow transient redistribution of calcium, sodium and potassium ions between cells and the medium leading to neuronal depolarization. Observations that Dyn A increases intracellular calcium ion concentration in rat cortical neurons through a non-opioid [22], non-glutamate receptor mediated mechanism [23], support this hypothesis. Acknowledgments: This work was supported by grants from the Swedish Research Council (to A.G. and G.B.), the European Commission (Grants HPRN-CT-2001-00242 and QLK3-CT-2002-01989) to A.G., the Swedish AFA Foundation to G.B. and The Wenner-Gren Foundations to V.V.

References [1] Fallon, J.H. and Leslie, F.M. (1986) Distribution of dynorphin and enkephalin peptides in the rat brain. J. Comp. Neurol. 249, 293–336. [2] Khachaturian, H., Watson, S.J., Lewis, M.E., Coy, D., Goldstein, A. and Akil, H. (1982) Dynorphin immunocytochemistry in the rat central nervous system. Peptides 3, 941–954. [3] Mansour, A., Fox, C.A., Burke, S., Meng, F., Thompson, R.C., Akil, H. and Watson, S.J. (1994) Mu, delta, and j-opioid receptor mRNA expression in the rat CNS: an in situ hybridization study. J. Comp. Neurol. 350, 412–438. [4] Pierce, J.P., Kurucz, O.S. and Milner, T.A. (1999) Morphometry of a peptidergic transmitter system: dynorphin B-like immunoreactivity in the rat hippocampal mossy fiber pathway before and after seizures. Hippocampus 9, 255–276. [5] Van Bockstaele, E.J., Sesack, S.R. and Pickel, V.M. (1994) Dynorphin-immunoreactive terminals in the rat nucleus accumbens: cellular sites for modulation of target neurons and interactions with catecholamine afferents. J. Comp. Neurol. 341, 1–15. [6] Fischli, W., Goldstein, A., Hunkapiller, M.W. and Hood, L.E. (1982) Isolation and amino acid sequence analysis of a 4000dalton dynorphin from porcine pituitary. Proc. Natl. Acad. Sci. USA 79, 5435–5437. [7] Xie, G.X. and Goldstein, A. (1987) Characterization of big dynorphins from rat brain and spinal cord. J. Neurosci. 7, 2049– 2055. [8] Day, R. and Akil, H. (1989) The posttranslational processing of prodynorphin in the rat anterior pituitary. Endocrinology 124, 2392–2405. [9] Costigan, M. and Woolf, C.J. (2002) No DREAM, No pain. Closing the spinal gate. Cell 108, 297–300. [10] De Vries, T.J. and Shippenberg, T.S. (2002) Neural systems underlying opiate addiction. J. Neurosci. 22, 3321–3325.

L. Hugonin et al. / FEBS Letters 580 (2006) 3201–3205 [11] Kreek, M.J., LaForge, K.S. and Butelman, E. (2002) Pharmacotherapy of addictions. Nat. Rev. Drug Discov. 1, 710–726. [12] Kuzmin, A., Madjid, N., Terenius, L., Ogren, S.O. and Bakalkin G. Big dynorphin, a prodynorphin-derived peptide produces NMDA receptor-mediated effects on memory, anxiolytic-like and locomotor behavior in mice. Neuropsychopharmacology (in press). [13] Trujillo, K.A. and Akil, H. (1991) Opioid and non-opioid behavioral actions of dynorphin A and the dynorphin analogue DAKLI. NIDA Res. Monogr. 105, 397–398. [14] Walker, J.M., Moises, H.C., Coy, D.H., Baldrighi, G. and Akil, H. (1982) Nonopiate effects of dynorphin and des-Tyr-dynorphin. Science 218, 1136–1138. [15] Hauser, K.F., Aldrich, J.V., Anderson, K.J., Bakalkin, G., Christie, M.J., Hall, E.D., Knapp, P.E., Scheff, S.W., Singh, I.N., Vissel, B., Woods, A.S., Yakovleva, T. and Shippenberg, T.S. (2005) Pathobiology of dynorphins in trauma and disease. Front. Biosci. 10, 216–235. [16] Vanderah, T.W., Laughlin, T., Lashbrook, J.M., Nichols, M.L., Wilcox, G.L., Ossipov, M.H., Malan Jr., T.P. and Porreca, F. (1996) Single intrathecal injections of dynorphin A or des-Tyrdynorphins produce long-lasting allodynia in rats: blockade by MK-801 but not naloxone. Pain 68, 275–281. [17] Wang, Z., Gardell, L.R., Ossipov, M.H., Vanderah, T.W., Brennan, M.B., Hochgeschwender, U., Hruby, V.J., Malan Jr., T.P., Lai, J. and Porreca, F. (2001) Pronociceptive actions of dynorphin maintain chronic neuropathic pain. J. Neurosci. 21, 1779–1786. [18] Xu, M., Petraschka, M., McLaughlin, J.P., Westenbroek, R.E., Caron, M.G., Lefkowitz, R.J., Czyzyk, T.A., Pintar, J.E., Terman, G.W. and Chavkin, C. (2004) Neuropathic pain activates the endogenous kappa opioid system in mouse spinal cord and induces opioid receptor tolerance. J. Neurosci. 24, 4576–4584. [19] Tan-No, K., Esashi, A., Nakagawasai, O., Niijima, F., Tadano, T., Sakurada, C., Sakurada, T., Bakalkin, G., Terenius, L. and Kisara, K. (2002) Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl-D -aspartate receptor mechanism. Brain Res. 952, 7–14. [20] Tan-No, K., Takahashi, H., Nakagawasai, O., Niijima, F., Sato, T., Sato, S., Sakurada, S., Marinova, Z., Yakovleva, T., Bakalkin, G., Terenius, L. and Tadano, T. (2005) A pronociceptive role of dynorphins in uninjured animals: nociceptive effects of N-ethylmaleimide mediated through inhibition of degradation of endogenous dynorphins. Pain 113, 301–309. [21] Singh, I.N., Goody, R.J., Goebel, S.M., Martin, K.M., Knapp, P.E., Marinova, Z., Hirschberg, D., Yakovleva, T., Bergman, T., Bakalkin, G. and Hauser, K.F. (2003) Dynorphin A (1–17) induces apoptosis in striatal neurons in vitro through alphaamino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor-mediated cytochrome c release and caspase-3 activation. Neuroscience 122, 1013–1023. [22] Hauser, K.F., Foldes, J.K. and Turbek, C.S. (1999) Dynorphin A (1–13) neurotoxicity in vitro: opioid and non-opioid mechanisms in mouse spinal cord neurons. Exp. Neurol. 160, 361–375. [23] Tang, Q., Lynch, R.M., Porreca, F. and Lai, J. (2000) Dynorphin A elicits an increase in intracellular calcium in cultured neurons via a non-opioid, non-NMDA mechanism. J. Neurophysiol. 83, 2610–2615. [24] Marinova, Z., Vukojevic´, V., Surcheva, S., Yakovleva, T., Cebers, G., Pasikova, N., Usynin, I., Hugonin, L., Fang, W., Hallberg, ¨ ., Hauser, K.F., M., Hirschberg, D., Bergman, T., Langel, U Pramanik, A., Aldrich, J.V., Gra¨slund, A., Terenius, L. and Bakalkin, G. (2005) Translocation of dynorphin neuropeptides across the plasma membrane. A putative mechanism of signal transmission. J. Biol. Chem. 280, 26360–26370. [25] Derossi, D., Joliot, A.H., Chassaing, G. and Prochiantz, A. (1994) The third helix of the Antennapedia homeodomain translocates through biological membranes. J. Biol. Chem. 269, 10444–10450. [26] Wadia, J.S., Stan, R.V. and Dowdy, S.F. (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 10, 310–315.

L. Hugonin et al. / FEBS Letters 580 (2006) 3201–3205 [27] Padari, K., Sa¨a¨lik, P., Hansen, M., Koppel, K., Raid, R., Langel, ¨ . and Pooga, M. (2005) Cell transduction pathways of U transportans. Bioconjugate Chem. 16, 1399–1410. [28] Deshayes, S., Morris, M.C., Divita, G. and Heitz, F. (2005) Cellpenetrating peptides: tools for intracellular delivery of therapeutics. Cell Mol. Life Sci. 62, 1839–1849. [29] Lundberg, P., Magzoub, M., Lindberg, M., Ha¨llbrink, M., ¨ . and Gra¨slund, A. Jarvet, J., Eriksson, L.E.G., Langel, U (2002) Cell membrane translocation of the N-terminal (1–28) part of the prion protein. Biochem. Biophys. Res. Commun. 299, 85–90. [30] Matsuzaki, K., Yoneyama, S., Murase, O. and Miyajima, K. (1996) Transbilayer transport of ions and lipids coupled with mastoparan X translocation. Biochemistry 35, 8450– 8456. [31] Pouny, Y., Papaport, D., Mor, A., Nicolas, P. and Shai, Y. (1992) Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes. Biochemistry 31, 12416–12423. [32] Derossi, D., Calvet, S., Tremblea, A., Brunisse, A., Chassaing, G. and Prochiantz, A. (1996) Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J. Biol. Chem. 271, 18188–18193. [33] Luo, D. and Saltzman, W.M. (2000) Synthetic DNA delivery systems. Nat. Biotechnol. 18, 33–37. [34] Gill, S.C. and von Hippel, P.H. (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319–326. [35] Chen, F.Y., Lee, M.T. and Huang, H.W. (2002) Sigmoidal concentration dependence of antimicrobial peptide activities: a case study on alamethicin. Biophys. J. 82, 908–914.

3205 [36] Fernandez-Carneado, J., Kogan, M.J., Pujals, S. and Giralt, E. (2004) Amphipathic peptides and drug delivery. Biopolymers 76, 196–203. [37] Zasloff, M. (2002) Antimicrobial peptides of multicellular organisms. Nature 415, 389–395. [38] Saara, K., Lindgrena, M., Hansena, M., Eirı´ksdo´ttira, E., Jianga, ¨ . (2005) Y., Rosenthal-Aizmana, K., Sassiana, M. and Langel, U Cell-penetrating peptides: a comparative membrane toxicity study. Anal. Biochem. 345, 55–65. [39] Tan-No, K., Cebers, G., Yakovleva, T., Hoon Goh, B., Gileva, I., Reznikov, K., Aguilar-Santelises, M., Hauser, K.F., Terenius, L. and Bakalkin, G. (2001) Cytotoxic effects of dynorphins through nonopioid intracellular mechanisms. Exp. Cell Res. 269, 54–63. [40] Matsuzaki, M., Yasuyuki, K., Akada, O., Murase, S., Yoneyama, M. and Zasloff, K. (1998) Mechanism of synergism between antimicrobial peptides magainin 2 and PGLa. Biochemistry 37, 15144–15153. [41] Magzoub, M., Oglecka, K., Pramanik, A., Eriksson, L.E.G. and Gra¨slund, A. (2005) Membrane perturbation effects of peptides derived from the N-termini of unprocessed prion proteins. Biochim. Biophys. Acta 1716, 126–136. [42] Arbuzova, A. and Schwarz, G. (1999) Pore-forming action of mastoparan peptides on liposomes: a quantitative analysis. Biochim. Biophys. Acta 1420, 139–152. [43] Matsuzaki, K., Yoneyama, S. and Miyajima, K. (1997) Pore formation and translocation of melittin. Biophys. J. 73, 831–838. [44] Benachir, T. and Lafleur, M. (1995) Study of vesicle leakage induced by melittin. Biochim. Biophys. Acta 1235, 452–460.