Abstract
In order to develop future therapeutic applications for cell penetrating peptides (CPPs), it is essential to characterize their internalization mechanisms, as they might affect the stability and the accessibility of the carried drug. Several internalization mechanisms have been described in literature, such as endocytosis and transduction. In this work we study the internalization mechanism in HeLa cells of two TIRAP derived peptides: pepTIRAP and pepTIRAPALA, where some of the cationic amino acids were replaced with alanines. Detailed analysis of internalization and the peptides electrostatic potential was carried out, to shed light on the internalization mechanism involved. Molecular modeling studies showed that the main difference identified between pepTIRAP and pepTIRAPALA is the distribution of their electrostatic potential field. The structure of pepTIRAP displays a predominantly positive potential when compared to pepTIRAPALA, which has a more balanced potential distribution. In addition, docking experiments show that interactions between pepTIRAP and negatively charged molecules on the cellular surface such as heparan sulfate are stronger than the ones exhibited by pepTIRAPALA. A mathematical model was proposed to quantify the amount of peptide internalized or non-specifically bound to the membrane. The model indicates a stronger interaction of pepTIRAP with the plasma membrane, compared to pepTIRAPALA. We propose these discrepancies are related to the differences in the electrostatic potential characteristics of each peptide. In the case of pepTIRAP, these interactions lead to the formation of nucleation zones, which are the first stage of the transduction internalization mechanism. These results should be considered for effective design of a cell penetrating peptide.
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Nori, A., K. D. Jensen, M. Tijerina, P. Kopeckova, and J. Kopecek (2003) Tat-conjugated synthetic macromolecules facilitate cytoplasmic drug delivery to human ovarian carcinoma cells. Bioconjug. Chem. 14: 44–50.
Choi, M., S. Rolle, M. Wellner, M. C. Cardoso, C. Scheidereit, F. C. Luft, and R. Kettritz (2003) Inhibition of NF-κB by a TATNEMO-binding domain peptide accelerates constitutive apoptosis and abrogates LPS-delayed neutrophil apoptosis. Blood 102: 2259–2267.
Fawell, S., J. Seery, Y. Daikh, C. Moore, L. L. Chen, B. Pepinsky, and J. Barsoum (1994) Tat-mediated delivery of heterologous proteins into cells. Proc. Natl. Acad. Sci. U. S. A. 91: 664–668.
Turner, J. J., G. D. Ivanova, B. Verbeure, D. Williams, A. A. Arzumanov, S. Abes, B. Lebleu, and M. J. Gait (2005) Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells. Nucleic Acids Res. 33: 6837–6849.
Lewin, M., N. Carlesso, C. H. Tung, X. W. Tang, D. Cory, D. T. Scadden, and R. Weissleder (2000) Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat. Biotechnol. 18: 410–414.
Joliot, A., C. Pernelle, H. Deagostini-Bazin, and A. Prochiantz (1991) Antennapedia homeobox peptide regulates neural morphogenesis. Proc. Natl. Acad. Sci. U. S. A. 88: 1864–1868.
Frankel, A. D. and C. O. Pabo (1988) Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55: 1189–1193.
Malecki, J., J. Wesche, C. S. Skjerpen, A. Wiedlocha, and S. Olsnes (2004) Translocation of FGF-1 and FGF-2 across vesicular membranes occurs during G1-phase by a common mechanism. Mol. Biol. Cell 15: 801–814.
Tunnemann, G., G. Ter-Avetisyan, R. M. Martin, M. Stockl, A. Herrmann, and M. C. Cardoso (2008) Live-cell analysis of cell penetration ability and toxicity of oligo-arginines. J. Pept. Sci. 14: 469–476.
Yoshikawa, T., T. Sugita, Y. Mukai, Y. Abe, S. Nakagawa, H. Kamada, S. Tsunoda, and Y. Tsutsumi (2009) The augmentation of intracellular delivery of peptide therapeutics by artificial protein transduction domains. Biomaterials 30: 3318–3323.
Deshayes, S., M. C. Morris, G. Divita, and F. Heitz (2005) Cell-penetrating peptides: tools for intracellular delivery of therapeutics. Cell. Mol. Life Sci. 62: 1839–1849.
Suzuki, T., S. Futaki, M. Niwa, S. Tanaka, K. Ueda, and Y. Sugiura (2002) Possible existence of common internalization mechanisms among arginine-rich peptides. J. Biol. Chem. 277: 2437–2443.
Vives, E., P. Brodin, and B. Lebleu (1997) A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 272: 16010–16017.
Derossi, D., S. Calvet, A. Trembleau, A. Brunissen, G. Chassaing, and A. Prochiantz (1996) Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J. Biol. Chem. 271: 18188–18193.
Richard, J. P., K. Melikov, H. Brooks, P. Prevot, B. Lebleu, and L. V. Chernomordik (2005) Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors. J. Biol. Chem. 280: 15300–15306.
Kaplan, I. M., J. S. Wadia, and S. F. Dowdy (2005) Cationic TAT peptide transduction domain enters cells by macropinocytosis. J. Control. Release 102: 247–253.
Console, S., C. Marty, C. Garcia-Echeverria, R. Schwendener, and K. Ballmer-Hofer (2003) Antennapedia and HIV transactivator of transcription (TAT) “protein transduction domains” promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans. J. Biol. Chem. 278: 35109–35114.
Fischer, R., K. Kohler, M. Fotin-Mleczek, and R. Brock (2004) A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides. J. Biol. Chem. 279: 12625–12635.
Duchardt, F., M. Fotin-Mleczek, H. Schwarz, R. Fischer, and R. Brock (2007) A comprehensive model for the cellular uptake of cationic cell-penetrating peptides. Traffic 8: 848–866.
Tunnemann, G., R. M. Martin, S. Haupt, C. Patsch, F. Edenhofer, and M. C. Cardoso (2006) Cargo-dependent mode of uptake and bioavailability of TAT-containing proteins and peptides in living cells. FASEB J. 20: 1775–1784.
Wadia, J. S., R. V. Stan, and S. F. Dowdy (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 10: 310–315.
Horng, T., G. M. Barton, R. A. Flavell, and R. Medzhitov (2002) The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors. Nature 420: 329–333.
Toshchakov, V. U., S. Basu, M. J. Fenton, and S. N. Vogel (2005) Differential involvement of BB loops of toll-IL-1 resistance (TIR) domain-containing adapter proteins in TLR4-versus TLR2-mediated signal transduction. J. Immunol. 175: 494–500.
Takeda, K., T. Kaisho, and S. Akira (2003) Toll-like receptors. Annu. Rev. Immunol. 21: 335–376.
Schnare, M., G. M. Barton, A. C. Holt, K. Takeda, S. Akira, and R. Medzhitov (2001) Toll-like receptors control activation of adaptive immune responses. Nat. Immunol. 2: 947–950.
Medzhitov, R., P. Preston-Hurlburt, E. Kopp, A. Stadlen, C. Chen, S. Ghosh, and C. A. Janeway Jr. (1998) MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways. Mol. Cell 2: 253–258.
Horng, T., G. M. Barton, and R. Medzhitov (2001) TIRAP: An adapter molecule in the Toll signaling pathway. Nat. Immunol. 2: 835–841.
Yamamoto, M., S. Sato, H. Hemmi, H. Sanjo, S. Uematsu, T. Kaisho, K. Hoshino, O. Takeuchi, M. Kobayashi, T. Fujita, K. Takeda, and S. Akira (2002) Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4. Nature 420: 324–329.
Low, W., A. Mortlock, L. Petrovska, T. Dottorini, G. Dougan, and A. Crisanti (2007) Functional cell permeable motifs within medically relevant proteins. J. Biotechnol. 129: 555–564.
Shi, J., T. L. Blundell, and K. Mizuguchi (2001) FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties. J. Mol. Biol. 310: 243–257.
Mizuguchi, K., C. M. Deane, T. L. Blundell, M. S. Johnson, and J. P. Overington (1998) JOY: Protein sequence-structure representation and analysis. Bioinformatics 14: 617–623.
Bernstein, F. C., T. F. Koetzle, G. J. Williams, E. F. Meyer Jr., M. D. Brice, J. R. Rodgers, O. Kennard, T. Shimanouchi, and M. Tasumi (1977) The Protein Data Bank: A computer-based archival file for macromolecular structures. J. Mol. Biol. 112: 535–542.
Eswar, N., B. Webb, M. A. Marti-Renom, M. S. Madhusudhan, D. Eramian, M. Y. Shen, U. Pieper, and A. Sali (2006) Comparative protein structure modeling using Modeller. Curr. Protoc. Bioinformatics Chapter 5: Unit 5.6.
Marti-Renom, M. A., A. C. Stuart, A. Fiser, R. Sanchez, F. Melo, and A. Sali (2000) Comparative protein structure modeling of genes and genomes. Annu. Rev. Biophys. Biomol. Struct. 29: 291–325.
Fiser, A., R. K. Do, and A. Sali (2000) Modeling of loops in protein structures. Protein Sci. 9: 1753–1773.
Sali, A. and T. L. Blundell (1993) Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234: 779–815.
Willard, L., A. Ranjan, H. Zhang, H. Monzavi, R. F. Boyko, B. D. Sykes, and D. S. Wishart (2003) VADAR: A web server for quantitative evaluation of protein structure quality. Nucleic Acids Res. 31: 3316–3319.
Davis, I. W., A. Leaver-Fay, V. B. Chen, J. N. Block, G. J. Kapral, X. Wang, L. W. Murray, W. B. Arendall 3rd, J. Snoeyink, J. S. Richardson, and D. C. Richardson (2007) MolProbity: All-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35: 375–383.
Dolinsky, T. J., J. E. Nielsen, J. A. McCammon, and N. A. Baker (2004) PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Res. 32: 665–667.
Baker, N. A., D. Sept, S. Joseph, M. J. Holst, and J. A. McCammon (2001) Electrostatics of nanosystems: Application to microtubules and the ribosome. Proc. Natl. Acad. Sci. U. S. A. 98: 10037–10041.
Schrödinger, L. L. C. (2010) The PyMOL molecular graphics system, version 1.3r1.
Bank, R. E. and M. Holst (2003) A new paradigm for parallel adaptive meshing algorithms. SIAM Review 45: 291–323.
Holst, M. (2001) Adaptive numerical treatment of elliptic systems on manifolds. Adv. Comput. Math. 15: 139–191.
Shaya, D., W. Zhao, M. Garron, Z. Xiao, Q. Cui, Z. Zhang, T. Sulea, R. Linhardt, and M. Cygler (2010) Catalytic mechanism of heparinase II investigated by site-directed mutagenesis and the crystal structure with its substrate J. Biol. Chem. 285: 20051–20061.
Frisch, M. J., G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, V. G. Zakrzewski, J. A. Montgomery, R. E. Stratmann, J. C. Burant, S. Dapprich, J. M. Millam, A. D. Daniels, K. N. Kudin, M. C. Strain, O. Farkas, J. Tomasi, V. Barone, M. Cossi, R. Cammi, B. Mennucci, C. Pomelli, C. Adamo, S. Clifford, J. Ochterski, G. A. Petersson, P. Y. Ayala, Q. Cui, K. Morokuma, D. K. Malick, A. D. Rabuck, K. Raghavachari, J. B. Foresman, J. Cioslowski, J. V. Ortiz, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. Gomperts, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara, C. Gonzalez, M. Challacombe, P. M.W. Gill, B. G. Johnson, W. Chen, M.W. Wong, J. L. Andres, M. Head-Gordon, E. S. Replogle, and J. A. Pople (1998) Gaussian 98 (Revision A.1) — Gaussian, Inc., Pittsburgh, PA.
Sanner, M. (1999) Python: A Programming Language for Software Integration and Development J. Mol. Graphics Mod. 17: 57–61.
Morris, G., R. Huey, W. Lindstrom, M. Sanner, R. Belew, D. Goodsell, and A. Olson (2009) AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility J. Comput. Chem. 30: 2785–2791.
Patel, L. N., J. L. Zaro, and W. C. Shen (2007) Cell penetrating peptides: intracellular pathways and pharmaceutical perspectives. Pharm. Res. 24: 1977–1992.
Rothbard, J. B., T. C. Jessop, and P. A. Wender (2005) Adaptive translocation: the role of hydrogen bonding and membrane potential in the uptake of guanidinium-rich transporters into cells. Adv. Drug. Deliv. Rev. 57: 495–504.
Wender, P. A., D. J. Mitchell, K. Pattabiraman, E. T. Pelkey, L. Steinman, and J. B. Rothbard (2000) The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: Peptoid molecular transporters. Proc. Natl. Acad. Sci. U. S. A. 97: 13003–13008.
Alon, R., E. A. Bayer, and M. Wilchek (1990) Streptavidin contains an RYD sequence which mimics the RGD receptor domain of fibronectin. Biochem. Biophys. Res. Commun. 170: 1236–1241.
Alon, R., E. A. Bayer, and M. Wilchek (1992) Cell-adhesive properties of streptavidin are mediated by the exposure of an RGD-like RYD site. Eur. J. Cell Biol. 58: 271–279.
Alon, R., E. A. Bayer, and M. Wilchek (1993) Cell adhesion to streptavidin via RGD-dependent integrins. Eur. J. Cell. Biol. 60: 1–11.
Hollinshead, M., J. Sanderson, and D. J. Vaux (1997) Anti-biotin antibodies offer superior organelle-specific labeling of mitochondria over avidin or streptavidin. J. Histochem. Cytochem. 45: 1053–1057.
Rodriguez-Melendez, R. (2000) Importance of biotin metabolism. Rev. Invest. Clin. 52: 194–199.
Lehninger, A. L., D. L. Nelson, and M. M. Cox (2000) Lehninger principles of biochemistry. 3rd ed. Worth Publishers, NY, USA.
Vives, E. (2003) Cellular uptake of the Tat peptide: An endocytosis mechanism following ionic interactions. J. Mol. Recognit. 16: 265–271.
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Flores, K.A., Salgado, J.C., Zapata-Torres, G. et al. Effect of the electrostatic potential on the internalization mechanism of cell penetrating peptides derived from TIRAP. Biotechnol Bioproc E 17, 485–499 (2012). https://doi.org/10.1007/s12257-011-0482-z
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DOI: https://doi.org/10.1007/s12257-011-0482-z