User:Fnb3

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The pHLIP peptide (pH (Low) Insertion Peptide) helix C of the bacteriorhodopsin protein.

applications and uses[edit]

Cell penetrating peptides (CPPs) are short peptides that facilitate cellular uptake of various molecular cargo (from small chemical molecules to nanosize particles and large fragments of DNA). The "cargo" is associated with the peptides either through chemical linkage via covalent bonds or through non-covalent interactions. The function of the CPPs are to deliver the cargo into cells, a process that commonly occurs through endocytosis with the cargo delivered to the endosomes of living mammalian cells.

CPPs hold great potential as in vitro and in vivo delivery vectors for use in research and medicine. Current use is limited by a lack of cell specificity in CPP mediated cargo delivery and insufficient understanding of the modes of their uptake.

CPPs typically have an amino acid composition containing either a high relative abundance of positively charged amino acids such as lysine or arginine, or have sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively.

The first CPP was discovered independently by two laboratories in 1988 when it was found that the trans-activating transcriptional activator (Tat) from Human Immunodeficiency Virus 1 (HIV-1) could be efficiently taken up from the surrounding media by numerous cell types in culture.[1] Since then, the number of known CPPs has expanded considerably and small molecule synthetic analogues with more effective protein transduction properties have been generated.[2]

Mechanisms of membrane translocation[edit]

Cell Penetrating peptides are of different sizes, amino acid sequences, and charges but all CPPs have one distinct characteristic, which is the ability to translocate the plasma membrane and facilitate the delivery of various different molecular cargoes to the cytoplasm or an organelle. There has been no real consensus as to the mechanism of CPP translocation but the theories of CPP translocation can be classified into three main entry mechanisms; direct penetration in the membrane, endocytosis-mediated entry, and translocation through the formation of a transitory structure. CPP transduction is an area of ongoing research. [3] [4]

Cell penetrating peptides (CPP) are able to transport different types of cargo molecules across plasma membrane, thus they act as molecular delivery vehicles. Cell penetrating peptides have found numerous applications in medicine as drug delivery agents in the treatment of different diseases including cancer, virus inhibitors, as well as contrast agents for cell labeling. Examples of the latter include acting as a carrier for GFP, MRI contrast agents, or quantum dots. [5]

Direct Penetration[edit]

File:PHLIP three state scheme.tif
Scheme showing pHLIP in solution, attached to the membrane, and inserted as a tramsmebrane helix, and the thermodyamic parameters of the transitions

The majority of early research suggested that the translocation of polycationic CPPs across biological membranes occurred via an energy independent cellular process. It was believed that translocation could progress at 4oC and most likely involved a direct electrostatic interaction with negatively charged phospholipids. Researchers proposed several models in attempts to elucidate the biophysical mechanism of this energy independent process. Although CPPs promote direct effects on the biophysical properties of pure membrane systems, the identification of fixation artifacts when using fluorescent labeled probe CPPs caused a reevaluation of CPP-import mechanisms.[6] These studies promoted endocytosis as the translocation pathway. An example of direct penetration has been proposed for Tat. The first step in this proposed model is an interaction with the unfolded fusion protein (TaT) and the membrane through electrostatic interactions which disrupt the membrane enough to allow the fusion protein to cross the membrane. After internalization the fusion protein refolds due the chaperon system. This mechanism was not agreed upon and other mechanisms involving clathrin-dependent endocytosis have been suggested. [7][8]
Recently a detailed model for direct translocation across the plasma membrane has been proposed [9][10]. This mechanism involves strong interactions between cell-penetrating peptides and the phosphate groups on both sides of the lipid bilayer, the insertion of charged side chains that nucleate the formation of a transient pore, followed by the translocation of cell-penetrating peptides by diffusing on the pore surface. This mechanism explains how key ingredients, such as the cooperativity among the peptides, the large positive charge, and specifically the guanidinium groups, contribute to the uptake. The proposed mechanism also illustrates the importance of membrane fluctuations. Indeed, mechanisms that involve large fluctuations of the membrane structure, such as transient pores and the insertion of charged amino acid side chains, may be common and perhaps central to the functions of many membrane protein functions. This model contains several controversial features, may be the most striking one is the formation of transient pores that facilitate the diffusion of the peptides across either the plasma membrane or the endosomal vesicles towards the cytosol. Recent experimental data has validated this key ingredient of the model showing that cell-penetrating peptides indeed form transient pores on lipid bilayers and on live cells[11].

Endocytosis mediated Translocation[edit]

Types Endocytosis Mediated by Cell Penetrating Peptides

Endocytosis is the second mechanism liable for cellular internalization. Endocytosis is the process of cellular ingestion by which the plasma membrane folds inward to bring substances into the cell. During this process cells absorb material from the outside of the cell by imbibing it with their cell membrane. The classification of cellular localization using fluorescence or by endocytosis inhibitors is the basis of most examination. Regrettably, the procedure used during preparation of these samples creates questionable information regarding endocytosis. Moreover, studies show that cellular entry of penetratin by endocytosis is an energy dependent process. This process is initiated by polyarginines interacting with heperan sulphates which promote endocytosis. Research has shown that TAT is internalized through a form of endocytosis called macropinocytosis.[12][13]

Studies have illustrated that endocytosis is involved in the internalization of CPPs but it has been suggested that different mechanisms could transpire at the same time. This is established by the behavior reported for penetratin and transportan wherein both membrane translocation and endocytosis, occur concurrently.[14][15]

Translocation Through the Formation of a Transitory Structure[edit]

Translocation Mediated by the Formation of Inverted Micelles

The third mechanism responsible for the translocation is based on the formation of the inverted micelles. Inverted micelles are aggregates of colloidal surfactants in which the polar groups are concentrated in the interior and the lipophilic groups extend outward into the solvent. According to this model, a penetratin dimer combines with the negatively charged phospholipids, thus generating the formation of an inverted micelle inside of the lipid bilayer. The structure of the inverted micelles permits the peptide to remain in a hydrophilic environment.[16][17] [18] Nonetheless, this mechanism is still a matter of discussion, because the distribution of the penetratin between the inner and outer membrane is non-symmetric. This non-symmetric distribution produces an electrical field that has been well established. Increasing the amount of peptide on the outer leaflets causes the electric field to reach a critical value that can generate an electroporation-like event.

The last mechanism implied that internalization occurs by peptides which belong to the family of primary amphipathic peptides, MPG and Pep-1. Two very similar models have been proposed based on physicochemical studies, consisting of circular dichroism, Fourier transform infrared, and nuclear magnetic resonance spectroscopy. These models are associated with electrophysiological measurements and investigations which have the ability to mimic model membranes such as monolayer at the air-water interface. The structure giving rise to the pores is the major difference between the proposed MPG and Pep-1 model. In the MPG model, the pore is formed by a b-barrel structure, whereas the Pep-1 is associated with helices. Additionally, strong hydrophobic phospholipid-peptide interactions have been discovered in both models. [19][20] In the two peptide models, the folded parts of the carrier molecule correlate to the hydrophobic domain, although the rest of the molecule remains unstructured. [21]

Translocation Mediated by a Transitory Structure

Cell penetrating peptide facilitated translocation is a topic of great debate. Evidence has been presented that translocation could use several different pathways for uptake. In addition the mechanism of translocation can be dependent on whether the peptide is free or attached to cargo. The quantitative uptake of free or CPP connected to cargo can differ greatly but studies haven’t proven if this change is a result of translocation efficiency or the difference in translocation pathway. It is probable that the results indicate that several CPP mechanisms are in competition and that several pathways contribute to CPP internalization. [22]


Magnetic resonance imaging[edit]

Examples of metal chelates successfully delivered into cells

Magnetic resonance imaging (MRI) is a powerful tool for disease diagnosis such as cancer metastasis and inflammation using different metal chelates. Metal chelates increase the contrast signal between normal and diseased tissues by changing the nuclear relaxation times of water molecules in their proximities. Typical examples are Gd3+ low molecular weight chelates, and superparamagnetic iron oxide (SPIO). In vivo administration of these agents allows the label of tumor cells; or cells can be labeled in vitro with contrast agents and then they can be injected and monitored in vivo by using MRI techniques. [23] [24] [25] SPIO nanoparticles confer high sensitivity in MRI but they have lower affinity for cells, they work at high concentrations. Functionalizations of these compounds using dentrimeric guanidines showed similar activities as TAT based CPPs but higher toxicity. New substrates based on dendrons with hydroxyl or amine peripheries show low toxicity. Applications of SPIO includes cell labeling in vivo, due to low toxicity, they are clinically approved for use in liver, spleen and gastrointestinal imaging. [26] The presence of octamer arginine residues allows cell membrane transduction of various cargo molecules including peptides, DNA, siRNA and contrast agents. However the ability of cross membrane is not unidirectional, arginie based CPPs are able to enter-exit the cell membrane displaying an overall decreasing of concentration of contrast agent and a decrease of magnetic resonance (MR) signal in time, this limits their application in vivo. To solve this problem, contrast agents with disulfide reversible bond between metal chelate and transduction moiety enhance the cell-associated retention. The disulfide bond is reduced by the target cell environment and metal chelate remains trapped into cytoplasm increasing the retention time of chelate into the target cell. [27] [28] [29] [30]

References[edit]

  1. ^ Protein Transduction: Cell Penetrating Peptides and Their Therapeutic Applications; Wagstaff, Kylie M.; Jans, David A; Current Medicinal Chemistry, Volume 13, Number 12, May 2006 , pp. 1371-1387(17)
  2. ^ Okuyama, M. and Laman, H. and Kingsbury, S.R. and Visintin, C. and Leo, E. and Eward, K.L. and Stoeber, K. and Boshoff, C. and Williams, G.H. and Selwood, D.L. (2007) Small-molecule mimics of an α-helix for efficient transport of proteins into cells. Nature Methods, 4 (2). pp. 153-159
  3. ^ . Opalinska, J. B.; Gewirtz, A. M. Nucleic-acid therapeutics: basic principles and recent applications. Nat. Rev. Drug Discov. 2002, 1, 503-514
  4. ^ Eckstein, F. The versatility of oligonucleotides as potential therapeutics. Expert. Opin. Biol. Ther. 2007, 7, 1021-1034.
  5. ^ Stewart, K., M., Hortonb, K., L., O. Kelley S., O., Org. Biomol. Chem., 2008, 6, 2242–2255
  6. ^ Luo, D.; Saltzman, W. M. Synthetic DNA delivery systems. Nat. Biotechnol. 2000, 18, 33-37.
  7. ^ Vives, E., Brodin, P. and Lebleu, B. (1997) J. Biol. Chem. 272, 16010–16017
  8. ^ Zelphati O. and Szoka F. C. Jr (1996) Intracellular distribution and mechanism of delivery of oligonucleotides mediated by cationic lipids. Pharm. Res. 13: 1367–1372
  9. ^ H. D. Herce & A. E. Garcia (2007) PNAS, 104, 20805 Download
  10. ^ H. D. Herce & A. E. Garcia (2008) Journal of Biological Physics, 33, 345
  11. ^ H. D. Herce et al (2009) Biophysical Journal, 97, 1917-1925 arXiv:0910.1736v1
  12. ^ Frankel, A. D.; Pabo, C. O. Cellular uptake of the tat protein from human immunodeficiency virus. Cell 1988, 55, 1189-1193
  13. ^ Lundberg M. and Johansson M. (2001) Is VP22 nuclear homing an artifact? Nat. Biotechnol. 19: 713–714
  14. ^ Lundberg M., Wikstrom S. and Johansson M. (2003) Cell surface adherence and endocytosis of protein transduction domains. Mol. Ther. 8: 143–150
  15. ^ J. Howl, I.D. Nicholl, Biochem. Soc. Trans (2007) 35, 767-769
  16. ^ Plenat T., Deshayes S., Boichot S., Milhiet P. E., Cole R., Heitz F. et al. (2004) Interaction of primary amphipathic cellpenetrating peptides with phospholipid-supported monolayers. Langmuir 20: 9255–9261
  17. ^ Deshayes S., Gerbal-Chaloin S., Morris M. C., Aldrian-Herrada G., Charnet P., Divita G. et al. (2004) On the mechanism of non-endosomial peptide-mediated cellular delivery of nucleic acids. Biochim. Biophys. Acta 1667: 141–147
  18. ^ Deshayes S., Heitz A., Morris M. C., Charnet P., Divita G. and Heitz F. (2004) Biochemistry 43: 1449–1457
  19. ^ Magzoub M., Kilk K., Eriksson L. E., Langel U. and Graslund A. (2001) Interaction and structure induction of cell-penetrating peptides in the presence of phospholipid vesicles. Biochim. Biophys. Acta 1512: 77–89
  20. ^ Deshayes S., Plenat, T., Aldran-Herrada, G., Divita G., Le Grimellec C. and Heitz F. (2004) Primary amphipathic cell penetrating peptides: structural requirements and interactions with model membranes. Biochemistry 43: 7698–7706
  21. ^ Derossi D., Calvet S., Trembleau A., Brunissen 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
  22. ^ Tilstra, J., Rehman, K.K., Hennon, T., Plevy, S.E., Clemens, P. and Robbins, P.D. (2007) Biochem. Soc. Trans. 35, 811–815
  23. ^ Lewis, B. K., Zywicke, H., Miller, B., van Gelderen, P., Moskowitz, B. M., Duncan, I. D., and Frank, J. A., Nat. Biotechnol. 2001(19), 1141–1147.
  24. ^ Pittet, M. J., Swirski, P. K., Reynolds, F., Josephson, L., and Weissleder, R., Nat. Protoc. 2006(1), 73–78.
  25. ^ Foster, P. J., Dunn, E. A., Karl, K. E., Snir, J. A., Nycz, C. M., Harvey, A. J., and Pettis, R. J., Neoplasia 2008(10), 207–216.
  26. ^ Martin, A., L., Bernas, L., M., Rutt, B., K., Foster, P., J., Gillies, E., R., Bioconjugate Chem. 2008(19), 2375–2384.
  27. ^ Allen, M. J., MacRenaris, K. W., Venkatasubramanian, P. N., and Meade, T. J. (2004) Chem. Biol. 2004(11), 301–307.
  28. ^ Futaki, S., Adv. Drug Delivery Rev. 2005(57) 547–558.
  29. ^ Futaki, S., Suzuki, T., Ohashi, W., Yagami, T., Tanaka, S., Ueda, K., and Sugiura, Y. (2001) J. Biol. Chem. 2001(276), 5836–5840.
  30. ^ Endres, P., J., MacRenaris,K., W., Stefan Vogt, S., Meade, T., J., Bioconjugate Chem. 2008(19), 2049–2059.


(1) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1965453/?tool=pubmed

(2) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2556629/?tool=pubmed
http://opa.yale.edu/news/article.aspx?id=2157

External links[edit]

See also[edit]

Category:Peptides Category:Cell biology Category:Biophysics Category:Oncology

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