Peptides Tales of Peptides Proceedings of the Thirty-First European Peptide Symposium. September 5-9, 2010, Copenhagen, Denmark

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2 Peptides 21 Tales of Peptides Proceedings of the Thirty-First European Peptide Symposium September 5-9, 21, Copenhagen, Denmark Edited by Michal Lebl Prompt Scientific Publishing, San Diego, CA, USA Morten Meldal Carlsberg Laboratory, Copenhagen, Denmark Knud J. Jensen University of Copenhagen, Faculty of Life Sciences, Denmark Thomas Høeg-Jensen Novo Nordisk A/S, Maaloev, Denmark European Peptide Society

3 ISBN Published by the European Peptide Society Sold and distributed by Copyright 21 the European Peptide Society All rights reserved. No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording or by any information storage and retrieval system, without written permission from the copyright owner. Produced by Prompt Scientific Publishing, San Diego, U.S.A.

4 Proceedings of the 31 st European Peptide Symposium Michal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors) European Peptide Society, 21 Selective Membrane Interactions of Nucleolar-Targeting Peptides Margarida Rodrigues 1, Gandhi Rádis-Baptista 2,3, Beatriz G. de la Torre 2, Miguel Castanho 1, David Andreu 2, and Nuno C. Santos 1 1 Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, , Portugal; 2 Departament de Ciències Experimentals I de la Salut, Universitat Pompeu Fabra, Barcelona, E-83, Spain; 3 Laboratório de Bioquímica e Biotecnologia, Instituto de Ciências do Mar, Universidade Federal do Ceará, Fortaleza, , Brazil Introduction Crotamine is one of the major components of the venom of Crotalus durissus terrificus, a rattlesnake from South America. This toxin, when present at high (mm) concentrations, leads to the spastic paralysis of the hind limbs [1]. However, when present at (μm) concentrations, it can selectively translocate into actively proliferating cells [2], both in vivo and in vitro and localize to the nucleus. Moreover, it was observed that crotamine is able to deliver specific cargoes into the interior of the cell [2,3]. These characteristics allowed its classification as cell-penetrating peptide (CPP). Nucleolar-targeting peptides (NrTP) were designed by structural dissection of crotamine and, at μm concentration, they were also able to penetrate different cell types and exhibit exquisite nucleolar localization [4]. This new family of peptides was, therefore, classified as novel CPP. The peptides used throughout this work were: NrTP1 (YKQCHKKGGKKGSG), NrTP2 (YKQCHKKGG- Ahx-KKGSG), NrTP5 (ykqchkkggkkgsg) and NrTP6 (KQSHKKGGKKGSG). Fluorescent derivatives of all peptides were also produced using rhodamine B as the fluorescent probe. The aim of this work was to pursue the study of NrTP molecular mechanism for translocation into cells, as well as to determine the ability of NrTP to deliver large molecules into cells. Results and Discussion The biophysical characterization was done by fluorescence spectroscopy using tyrosine intrinsic fluorescence as well as rhodamine B labeled NrTP. The work included quenching studies, quantification of partition into membrane model systems and translocation experiments. Quenching experiments with acrylamide showed a linear dependence on the acrylamide concentration. This result indicates that Tyr residues are exposed to acrylamide and that there is no peptide aggregation in solution. The Stern-Volmer constant (K SV ) for NrTP1 is 9.7 ±.2 M -1, for NrTP ±.3 M -1 and for free Tyr 28.1 ±.2 M -1. In the presence of lipid vesicles, the K SV has a three-fold decrease except for free Tyr. This can be interpreted as a reduction of Tyr residues exposure to acrylamide. NrTP1 and NrTP2 interact with lipid membranes; consequently, they become less accessible to the quencher. NrTP1 and NrTP2 (Table 1) showed higher partition coefficients (for review see [5]) for POPC (zwitterionic) and POPG (anionic), both liquid state phospholipids, than for POPC:cholesterol (raft-like mixture on the liquid ordered state). For the translocation experiments, rhodamine B labeled NrTP were used and tested with giant multilamellar, NBD-labeled vesicles. The co-localization of fluorescence spikes from NBD and rhodamine B (NrTP1-RhB) (Figure 1-A) indicates successful peptide translocation once it represents the presence of peptide in the inner membranes that appear Table 1. Partition coefficient (K p ) for NrTP1 and NrTP2 in POPC, POPC:Chol and POPG LUV Lipid NrTP1 NrTP2 K p (x 1 3 ) ± SE K p (x 1 3 ) ± SE POPC 2.7 ± ±.8 POPC:Chol (2:1) 1.2 ±.2.7 ±.1 POPG 2.9 ± ±.3 inside some of the lipid vesicles. Despite the fact that the peptide fluorescence intensity is much lower than the one from NBD labeled vesicles, it is significantly different from the background, which thus, validates the conclusions. On the other hand, the translocation in cells is clearly more efficient 48

5 (Figure 1 B). Both lymphocyte cell lines (Bv173 and MOLT4) and peripheral blood mononuclear cells (PBMC) showed very high levels of peptide entry. These experiments were done using both NrTP1-RhB and NrTP5-RhB, and the results are very similar both for the profile in multilamellar giant vesicles and cells. A B 2 2 Fluorescence / AU Fluorescence / AU distance / μm distance / μm Fig. 1. Translocation of NrTP1-RhB into giant multilamellar vesicles (POPC + DPPE- NBD 1%) and Bv173 (pre-b cell leukemia) cell line. Graphs represent fluorescence intensity along a longitudinal line drawn on the vesicle or cell, respectively. Panel A shows the co-localization of NBD (---) (488 nm laser) with NrTP1-RhB 15 mm ( ) (561 nm laser). Each spike of NBD fluorescence corresponds to a lipid bilayer. Panel B shows the co-localization of the nuclear dye Hoeschst (---) (45 nm laser) with NrTP1-RhB 15 mm ( ), (561nm laser). Finally, a conjugate of NrTP (NrTP6-C) bound to β galactosidase (from E. coli) was prepared by chemical synthesis. This conjugate maintains enzymatic activity and is stable at 4ºC for several days, retaining its activity after -2ºC storage. Internalization studies for the delivery of β-galactosidase into HeLa cells were conducted with the above mentioned conjugate. Efficient translocation of the enzyme was detected in a cell free extract fluorescence based assay (Figure 2). Fluorescence / a.u. 44 nm time / min The work done so far with this new family of CPP has revealed strong interaction and translocation with lipid model systems. Moreover, and as a proof of concept that these cellpenetrating peptides are good carriers for the delivery of large molecules into the cell interior, we have successfully observed that NrTP can translocate β-galactosidase into cells. Acknowledgments Partial funding and M.R. PhD grant (SFRH/BD/37432/27) by Portuguese Ministry of Science (FCT- MCTES) and by Spanish Ministry of Science and Innovation (MICINN, grant BIO CO3) are acknowledged. References 1. Nicastro, G. Eur. J. Biochem. 27, (23). 2. Kerkis, A., et al. FASEB J. 18, (24). 3. Nascimento, F.D., et al. J. Biol. Chem. 282, (27). 4. Rádis-Baptista, G., et al. J. Med. Chem. 51, (28). 5. Santos, N.C., et al. Biochim. Biophys. Acta. 1612, (23). Fig. 2. Progression curves of β-galactosidase enzymatic activity. Fluorescence intensity is measured at 44 nm upon addition of enzyme to.5 mm of MUG. The plot represents the in vitro activity of the conjugate (NrTP6-C-βgalactosidase) when it is present at.5 nm ( ), 2 nm ( ), 5 nm ( ) and 7 nm ( ).The progression curve of a cell free extract resulting from the incubation of.3 mm of conjugate with HeLa cells ( ). 49