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Cell penetrating peptides.
Cell penetrating peptides (CPPs) are a class of short amino acid sequences which are sufficient for crossing cell membranes and delivering themselves along with any attached cargo into the cytoplasm of cells. They were branded CPPs recently, while the term "protein transduction domain" (PTD) was used in the early days of their study, when the necessary sequences were being isolated from native proteins with cell-penetrating activity (notably, HIV-1 Tat).
Most of the scientific literature I have found on CPPs focuses on their potential relevance for targeted drug delivery, and mostly discusses the delivery of proteins and nanoparticles via fusion of CPPs to the N-terminus of the target protein. The only This Site question & answer on CPPs also points to a discussion of N-terminal fusion of CPPs to a cargo peptide. However, research on cell penetrating peptides has come a long way since the 2005 article referenced in that post.
The 2012 Milletti review highlights that there has been a dramatic increase in the number of different classes of cell penetrating peptides, including hydrophobic CPPs, primary amphipathic CPPs, alpha-helical amphipathic CPPs, beta sheet amphipathic CPPs, and proline-rich CPPs, among others. And that is just between 2005 and 2012! The Milletti 2012 article also covers some CPPs isolated from other viruses.
I have not been able to find a review of similar detail written since, although I'm sure much progress has been made in the field.
Although it is not my main question, I would appreciate any citations for more recent reviews which go into detail on the molecular mechanism of cell penetration for one or more classes of CPPs - especially a review of the function of PTDs in their native proteins.
I have only begun to research CPPs and PTDs in their native proteins and in the drug-delivery setting because it became apparent that a protein I am studying for an undergraduate thesis may have an internal PTD which is essential for its function. I am no expert on the topic and I would appreciate information on CPPs/PTDs including further synonyms for CPPs/PTDs to aid in my literature search - especially synonyms specific to native proteins with CPP-like functions.
What are the secondary structure requirements for cell-penetrating peptides?
My question is whether the previously described positively charged CPPs and/or the new classes of CPPs described in Milletti 2012 are only functional as N-terminal fusions, or whether they typically function efficiently when they are incorporated within an internal loop (in terms of primary sequence, but still exposed to the solvent). The 2005 article cited in the previous This Site question on this topic stated that the CPP derived from HIV-1 Tat is based on residues 47-57, which are internal residues of the 86 amino acid protein - a protein which has cell-penetrating activity in its native role in HIV-1 infection.
Therefore, my question is not whether or not this ever occurs - it clearly does - but to what extent do CPPs isolated from internal sequences of native proteins have CPP-like functions in their native proteins?
The system I am studying for my thesis is a virus, so any information specifically on CPPs and PTDs function in the native proteins of viruses would be particularly useful.
Jones, S. W., Christison, R., Bundell, K., Voyce, C. J., Brockbank, S. M. V., Newham, P., & Lindsay, M. A. (2005). Characterisation of cell-penetrating peptide-mediated peptide delivery. British Journal of Pharmacology, 145(8), 1093-1102. http://doi.org/10.1038/sj.bjp.0706279
Milletti, F. (2012). Review: Cell-penetrating peptides: classes, origin, and current landscape. Drug Discovery Today, 17850-860. doi:10.1016/j.drudis.2012.03.002
Vivès, E., Brodin, P., & Lebleu, B. (1997). A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. The Journal Of Biological Chemistry, 272(25), 16010-16017.
If anyone interested in answering this question is not an expert, the best resource I can currently provide for a list of CPPs derived from natural proteins (besides the Milletti 2012 article above) is:
Hallbrink, M., Kilk, K., Elmquist, A., Lundberg, P., Lindgren, M., Jiang, Y., &… Langel, U. (n.d). Prediction of cell-penetrating peptides. International Journal Of Peptide Research And Therapeutics, 11(4), 249-259.
I will be continuing to research this topic for my thesis, so if you're interested in answering this question but require more information, feel free to comment asking for specific information that would help.
I am unable to give explanation to your question as a single answer but I am sure that below mentioned articles will clarify your doubts.
Cell-Penetrating Peptides: Design Strategies beyond Primary Structure and Amphipathicity - Molecules 2017, 22(11), 1929; doi:10.3390/molecules22111929. PMID: 29117144
Cell-penetrating peptides and their utility in genome function modifications (Review). Int J Mol Med. 2017 Dec;40(6):1615-1623. doi: 10.3892/ijmm.2017.3172. Epub 2017 Oct 4. PMID: 29039455
Quantitative fluorescence spectroscopy and flow cytometry analyses of cell-penetrating peptides internalization pathways: optimization, pitfalls, comparison with mass spectrometry quantification. Scientific Reports volume 6, Article number: 36938 (2016). doi:10.1038/srep36938
Cell-penetrating peptides: from molecular mechanisms to therapeutics
The recent discovery of new potent therapeutic molecules which do not reach the clinic due to poor delivery and low bioavailability have made the delivery of molecules a keystone in therapeutic development. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including CPPs (cell-penetrating peptides), which represent a new and innovative concept to bypass the problem of bioavailability of drugs. CPPs constitute very promising tools and have been successfully applied for in vivo. Two CPP strategies have been described to date the first one requires chemical linkage between the drug and the carrier for cellular drug internalization, and the second is based on the formation of stable complexes with drugs, depending on their chemical nature. The Pep and MPG families are short amphipathic peptides, which form stable nanoparticles with proteins and nucleic acids respectively. MPG- and Pep-based nanoparticles enter cells independently of the endosomal pathway and efficiently deliver cargoes, in a fully biologically active form, into a large variety of cell lines, as well as in animal models. This review focuses on the structure—function relationship of non-covalent MPG and Pep-1 strategies, and their requirement for cellular uptake of biomolecules and applications in cultured cells and animal models.
Cell-penetrating peptides (CPPs) can transport various cargoes through membranes of live cells. Since the first generations of CPPs suffered from insufficient cell and tissue selectivity, stability against proteases, and escape from endosomes, a new generation of peptides, with optimized properties, was developed. These are either derived from natural sources or created through the combination of multivalent structures. The second method allows achieving high internalization efficiency, high cell and tissue selectivity, and release from endosomes via hybrid structures, combining sequences for endosomal release, homing sequences, and sequences for activation at the target tissue and for local delivery of cargoes. CPPs with innate tumor selectivity include azurin, crotamine, maurocalcine, lycosin-I, buffalo cathelicidin, and peptide CB5005. Some of them can penetrate the membranes of live cells and influence intracellular signaling pathways, thereby exerting cytotoxic effects against tumor cells. To obtain multilayer penetration and stabilization against proteolytic degradation, as well as for better handling, CPPs are often conjugated to nanoparticles. A special problem for tumor treatment is the efficiency of drug transport through three-dimensional cell cultures. Therefore, the capability of CPPs to deliver the drug even to the innermost tissues is of crucial importance. Notably, the ability of certain CPPs to penetrate barriers such as skin, the blood-brain barrier (BBB), and cornea or conjunctiva of eyes enabled the replacement of dangerous and painful injections with soothing sprays, creams, and drops. However, it is difficult to rank the efficacy of CPPs because transport efficiency and tissue selectivity depend not only on the CPP itself but also on the target tissue or organ, as well as on the cargo and method of CPP-cargo coupling. Therefore, the present review describes some examples of new-generation CPPs and aims to provide advice on how to find or create the right CPP for a given task.
Chapter 10 - Peptide and Protein Delivery with Cell-penetrating Peptides
The most commonly used cell-penetrating peptides (CPPs): Tat, oligoarginine, and transportan, have all been demonstrated to facilitate the entry of protein/peptide cargoes into cells both in vitro and in vivo. In cellular systems, transportan has displayed greater internalization properties than the aforementioned arginine-rich CPPs and their uptake efficiencies can be depicted as follows: transportan > oligoarginine > Tat peptide. When picking the “right” CPP sequence for cargo delivery, both of these aspects need to be considered, and when lower concentrations are used, the transportan or oligoarginine should be preferred. In vivo, however, the uptake efficiency, specificity, and toxicity have not been extensively studied for the different CPPs. Nevertheless, it is evident that for targeted delivery, some extra motifs need to be added to the CPP sequence. A rather promising aspect for CPP-mediated delivery of peptides/proteins is that transcytosis in endothelial cells requires caveolin. Several CPPs, but especially transportan, exploit the caveolin pathway for cell entry, possibly giving transportan a beneficial “edge” in vivo. Despite the number of obstacles and pending challenges still faced today, the growing number of examples of in vivo delivery, Tat-HSP70 for neuronal rescue and Tat-Bcl-x(L) for improved neuronal precursor cell survival, confirm that the problems can be overcome in one way or another.
Why Use DDS?
The human organism poses many difficulties for the safe delivery of effective pharmaceuticals to the target cell 14 . Systemic administration and endovenous strategies allow for 100% bioavailability, obviating enteric, and hepatic metabolic degradation. Once in the bloodstream, the drug lead needs to overcome some challenges, avoid the immune system response (complement activation and phagocytosis), as well as serum protein aggregation and kidney filtration 24 . Once near the target cell, the eukaryotic membrane will pose further obstacles, being an impermeable barrier for most of xenobiotics 11, 15, 24, 25 . The plasma membrane only allows the influx of small compounds, requiring transporters for most hydrophilic macromolecules 17 .
The need of DDS for small molecules and gene-based tailored therapies also results from its biochemical characteristics such as poor stability, lack of cellular uptake and insufficient ability to reach targets 18, 19, 26, 27 . These are therefore the new key challenges in medicine – how to obtain effective therapeutic agents that act locally with limited adverse off-target effects 26 , with no loss of pharmaceutical potency or need for increasing dosages.
Several DDS have been developed 14, 28, 29 . While envisioning such technology, one should consider the use of biocompatible materials for simple, yet strong assembly processes. Optimization and fine-tuning of the biophysical-chemical parameters to enhance the DDS pharmacokinetic and pharmacodynamic properties are also urgent demands in this field 14, 26 . A successful DDS must work on different tissues, have fast endosome release, be functional at low dosage, have no toxicity, and be easy to administer concerning therapeutical applications 18 .
Several technologies have been developed and some examples of DDS approaches are: electroporation 30 , ultrasound mediated plasmid delivery 31 , viral delivery 32 , nebulization 33 , direct chemical modification 34 , and association with nonviral delivery vehicle such as lipids 35 , liposomes 36 , dendrimers 37 , cationic polymers 38 , inorganic particles 39 , carbon nanotubes 40 , small molecules 41 , receptor ligands 42 , more recently, supercharged proteins 43, 44 , and CPP 18, 45, 46 . This review will focus on one specific kind of DDS, CPP, mainly on its features and its application to clinical therapies. Figure 1 exemplifies the status of DDS technologies and the cargoes that were already delivered into cells using cellular endocytic pathways or by means of direct membrane translocation.
Application of CPP strategies to the delivery of therapeutic molecules
The number of applications using CPPs is consciously increasing, and so far more than 300 studies using either covalent or non-covalent CPP-based strategies from in vitro to in vivo have been reported ( Dietz and Bähr, 2004 Gros et al., 2006 Moschos et al., 2007 Patel et al., 2007 Foerg and Merkle, 2008 ). The interest for CPPs is mainly due to their low cytotoxicity and to the fact that there is no limitation for the type of cargo. Although CPPs have been used to improve delivery of cargo that varies greatly in size and nature (small molecules, oligonucleotide, plasmid DNA, peptide, protein, nanoparticle, lipid-based formulation, virus, quantum dots) most of the applications describe the delivery of oligopeptide/protein ( Dietz and Bähr, 2004 Gros et al., 2006 Patel et al., 2007 ) and nucleic acids or analogs ( Juliano et al., 2008 ) (Table 1).
CPP-based strategies for gene delivery
The poor permeability of the plasma membrane of eukaryotic cells to DNA together with the low efficiency of DNA or oligonucleotides to reach their target within cells constitutes the two major barriers for the development of these therapeutic molecules. In the last decade, a number of peptide carriers that combine DNA binding, mainly electrostatic domain (polylysine and polyarginine) and membrane-destabilizing properties have been developed to facilitate gene transfer into cultured cells and living animals ( Niidome and Huang, 2002 Glover et al., 2005 Morris et al., 2008 ). Amphipathic peptides with pH-dependent fusogenic and endosomolytic activities such as the fusion peptide of HA2 subunit of influenza hemaglutinin, or synthetic analogs GALA, KALA, JTS1, and histidine-rich peptides have been shown to increase transfection efficiency when associated with poly-L-lysine/DNA, condensing peptide/DNA, cationic lipids, poly-ethyleneimine or polyamidoamine cascade polymers (for review: Morris et al., 2008 ). Single peptide chains able to condense DNA and to favour endosomal escape (PPTG1) ( Rittner et al., 2002 ) or prevent endosomal uptake (MPG: Morris et al., 1999 ) have also been used for gene delivery in cultured cells. However, only a few CPPs have been validated in vivo for gene delivery and so far, the secondary amphipathic peptide PPTG1 constitutes one of the only examples reporting a significant in vivo gene expression response following intravenous injection ( Rittner et al., 2002 ). Tat, Transportan and polyarginine CPPs have been associated with other lipid-based non-viral gene delivery methods, including liposomes, PEI or nanostructures ( Branden et al., 1999 Tung et al., 2002 Ignatovich et al., 2003 Rudolph et al., 2003 Kilk et al., 2005 ). The association of Tat and octa-arginine to pharmaceutical nano-carriers, described as non-viral delivery systems based on new packing concepts ‘Programmed packaging’ Multifunctional Envelope-type NanoDevice (MEND) ( Kogure et al., 2004 MacKay et al., 2008 ), has been shown to improve gene delivery and to offer the advantage of combining delivery, packaging and targeting motifs within the same particle ( Torchilin, 2008 Vivès et al., 2008 ).
The second major barrier of non-viral gene delivery systems is their poor nuclear translocation, which is however essential for transfection of non-dividing cells and gene therapy. In order to improve nuclear delivery of DNA-plasmids, synthetic peptides containing NLS have been extensively applied ( Cartier and Reszka, 2002 Escriou et al., 2003 ). Most of these studies were performed with the sequence derived from SV40 large T antigen NLS (PKKKRKV). This sequence was associated with either membrane-penetrating or cationic peptides, but also directly linked to cargoes or combined with other transfection methods to facilitate delivery into the nucleus. Moreover, NLS sequences have been associated with different hydrophobic CPPs in order to favour nuclear targeting as well as DNA binding and compaction. The NLS domain of MPG has been shown to improve the nuclear translocation of nucleic acids without requiring nuclear membrane breakdown during mitosis. MPG technology has been applied to both plasmid DNA and oligonucleotide delivery with high efficiency into a large number of adherent and suspension cell lines ( Simeoni et al., 2003 Morris et al., 2007b ).
CPP-based strategies for oligonucleotide analog delivery
Steric block small neutral oligonucleotide including PNAs and phosphorodiamidate morphorodiamidate morpholino-oligomers (PMO) constitute potent molecules for either antisense application or mRNA splicing correction strategies. Several CPPs have been successfully applied for the delivery of uncharged PNA and PMO in vitro and in vivo through covalent coupling ( Gait, 2003 Moulton and Moulton, 2004 Zatsepin et al., 2005 Juliano et al., 2008 ). Originally reported with Transportan for in vivo delivery of an antisense PNA targeting galanine receptors and modifying pain transmission ( Pooga et al., 1998 ), the use of CPPs for steric block oligonucleotide delivery has been extended to Tat, penetratin, TP10 (a short version of Transportan) and arginine-rich peptides. Several CPP-based covalent approaches have been reported for the delivery of antisense PNA ( Koppelhus and Nielsen, 2003 ). However only a few have been used in vivo, and until recently none of them were reported to be active at submicromolar concentrations ( Gait, 2003 Abes et al., 2007 ). A detailed study of CPP-mediated PNA delivery has reported that the major limitation is due to their endosomal sequestration ( Koppelhus and Nielsen, 2003 ), and more recently new CPPs have been described by Lebleu and Gait groups including R6-penetratin and 6-aminohexanoic acid spaced oligoarginine [(R-Ahx-R)4], which exhibit limited endosomal sequestration and lead to submicromolar antisense or splicing correction response ( Abes et al., 2006 2007 ). These CPPs have been validated in vivo for splicing correction on two therapeutic models: Duchenne's muscular dystrophy ( Fletcher et al., 2007 ) and coronavirus replication in mice ( Burrer et al., 2007 ). Non-covalent strategies have also been applied to the delivery of PNA and DNA mimic molecules ( Nan et al., 2005 ). Pep-3 peptide, a variant of Pep-1, was successfully applied to the delivery of PNA and analogs targeting the cell cycle regulatory protein cyclin B1 in vitro and in vivo ( Morris et al., 2004b 2007b ). Interestingly, the nanoparticle organization of Pep-3/PNA complex allows functionalization of the surface layer of the particle, and PEGylation of the carrier significantly improves the efficacy of the response by stabilizing the complexes. This study shows that such a modification improves Pep-3 for systemic administration into mice, thereby allowing for a significant reduction of the dose required to induce a specific and robust biological response, which consequently limits non-specific cytotoxic effects described upon treatment with high concentrations of CPP-PNA conjugate or non-covalenty complexes ( Morris et al., 2007b ).
Oligonucleotide and siRNA delivery
Decoy oligonucleotides and short interfering RNAs (siRNA) constitute powerful biomedical tools to control protein activation and/or gene expression post-transcriptionally. ( Elbashir et al., 2001 Hannon, 2002 ). However, the major limitation of siRNA applications, like most antisense or nucleic acid-based strategies remains their poor cellular uptake associated with the poor permeability of the cell membrane to nucleic acids. Several viral and non-viral strategies have been proposed to improve the delivery of either siRNA-expressing vectors or synthetic siRNAs both in cultured cells and in vivo ( De Fougerolles et al., 2007 Juliano et al., 2008 ). CPP-based strategies have been developed to improve the delivery of oligonucleotides both in vitro and in vivo. Delivery of charged oligonucleotide and siRNA is more challenging as multiple anionic charges of the nucleic acid interact with CPP moiety and inhibit uptakes by steric hindrance. Delivery of charged oligonucleotide was achieved by using either peptide-based non-covalent or PNA-hybridization strategies. In the latter, CPPs are covalently linked to a PNA that is able to hybridize with a double-stranded decoy oligonucleotide containing on one strand a flanking sequence complementary to the PNA. Strategies have been applied with Transportan and TP10 CPP for the delivery of decoy oligonucleotide interacting with NFkB or Myc ( Fisher et al., 2004 El-Andaloussi et al., 2005 ). The MPG peptide-based delivery system has been successfully applied for the delivery of various type of nucleic acid, including phosphodiester-oligonucleotide targeting the protein phosphatase cdc25C ( Morris et al., 1999 ), phosphorothioate-oligonucleotides targeting MDR-1 promoter in human CEM leukaemia cells ( Marthinet et al., 2000 ) and thio-phosphoramidate telomerase template antagonists in cancer cells ( Asai et al., 2003 Gryaznov et al., 2003 ). Several CPP-based strategies have been used for the delivery of siRNA into cultured cells. siRNA covalently linked to Transportan ( Muratovska and Eccles, 2004 ) and penetratin ( Davidson et al., 2004 ) have been associated with a silencing response. Nevertheless, non-covalent strategies appear to be more appropriate for siRNA delivery and yield significant associated biological response ( Simeoni et al., 2003 Kim et al., 2006 Veldhoen et al., 2006 Crombez et al., 2007 Kumar et al., 2007 Lundberg et al., 2007 Meade and Dowdy, 2007 ). MPG peptide has been reported to improve siRNA delivery into a large panel of cell lines including adherent cell lines, cells in suspension, cancer and challenging primary cell lines ( Simeoni et al., 2003 Morris et al., 2004a Nguyen et al., 2006 ). MPG has been applied for in vivo delivery of siRNA targeting OCT-4 into mouse blastocytes ( Zeineddine et al., 2006 ) and of siRNA targeting an essential cell cycle protein, cyclin B1 intravenous injection of MPG/cyclin B1 siRNA particles has been shown to efficiently block tumour growth ( Crombez et al., 2007 ). A variant of MPG (MPG-alpha) harbouring five mutations in the hydrophobic domain, in order to favour helical conformation of the peptide, has also been shown to improve siRNA delivery ( Veldhoen et al., 2006 ). However, such modifications of MPG increase toxicity and favour endosomal cellular uptake ( Deshayes et al., 2004c Veldhoen et al., 2006 ). This non-covalent approach has been extended to other CPPs including polyarginine- ( Kim et al., 2006 Kumar et al., 2007 ), penetratin- ( Lundberg et al., 2007 ) and Tat- ( Meade and Dowdy, 2007 ) derived peptides. Tat peptide associated with an RNA-binding motif has been reported to block in vivo epidermal growth factor (EGF) factor, cholesterol-Arg9 has been shown to enhance siRNA delivery in vivo against vascular endothelial growth factors ( Kim et al., 2006 ) and more recently, a small peptide derived from rabies virus glycoprotein associated to polyarginine R9 has been shown to deliver siRNA in the CNS ( Kumar et al., 2007 ).
Over the past 5 years, a dramatic emergence of new potential therapeutic molecules has occurred, mainly due to the development of proteomics and genomics. However, these molecules still remain limited by their poor ability to enter cells. In order to render them more applicable for therapy in vivo, an increasing interest is being taken in the development of non-viral delivery methods (Niidome and Huang, 2002 Torchilin, 2005 ). For this aim, CPPs (cell-penetrating peptides) have been successfully used to improve the delivery of biologically active macromolecules, including nucleic acids, peptides and proteins, both in cell culture and in vivo (Järver and Langel, 2004 Gupta et al., 2005 ). Two major strategies have been developed. The first is based on natural or chimaeric PTDs (protein transduction domains) covalently linked to cargoes, the most representative of which include a peptide derived from the HIV-1 protein Tat (Fawell et al., 1994 Vivés et al., 1997 Frankel and Pabo, 1998 Schwarze et al., 1999 ), polyarginine (Wender et al., 2000 Futaki et al., 2001 ), the third helix of pAnt (antennapedia homeodomain protein) (Derossi et al., 1994 ) and transportan (Pooga et al., 1998 ). The second is based on primary amphiphatic peptides, such as MPG or Pep-1, which form stable non-covalent complexes with cargoes (Morris et al., 1997 , 2001 Simeoni et al., 2003 Gros et al., 2006 ). The cellular uptake mechanism of PTDs has been shown to be essentially associated with endosomal pathways (Richard et al., 2003 , 2005 Nakase et al., 2004 Wadia et al., 2004 ). However, clear evidence for distinct routes of cellular uptake have been reported, some of which are independent from the endosomal pathway and involve transmembrane potentials (Dom et al., 2003 Terrone et al., 2003 Thoren et al., 2003 Rothbard et al., 2004 Deshayes et al., 2005 Pujals et al., 2006 ).
The first contact that CPPs make with cells occurs through components of the extracellular matrix, the proteoglycans, which then trigger cellular uptake. Proteoglycans are heterogeneous proteins that carry one or more GAG (glycosaminoglycan) side chains, and that vary in size and shape (Kjellen and Lindahl, 1991 Esko and Selleck, 2002 ). Proteoglycans tend to form electrostatic interactions with molecules, which are primarily charge-mediated and dependent on the number of charges (Ruoslahti, 1998 ). Therefore they constitute a membrane ‘anchor’ through their GAG chains for a large variety of ligands (Sawitsky et al., 1996 Esclatine et al., 2001 Juliano, 2002 Couchman, 2003 ). Proteoglycans have been reported to be involved in different pathways controlling cell motility, shape or cell proliferation, which are directly associated with the dynamics of the cytoskeleton and actin network (Ruoslahti, 1988 Sawitsky et al., 1996 Esclatine et al., 2001 Juliano, 2002 Couchman, 2003 Beauvais and Rapraeger, 2004 Iozzo, 2005 ). The HSPGs (heparan sulfate proteoglycans) syndecan and glycan interact with the actin network via their cytoplasmic tail actin-binding protein and are both involved in the regulation of membrane ‘receptors’ and the cellular uptake of macromolecules (Couchman, 2003 Yoneda and Couchman, 2003 Beauvais and Rapraeger, 2004 ). GAG and HSPG play a central role in the translocation mechanism of polycationic carriers, liposomes (Mislick and Baldeschwieler, 1996 Kopatz et al., 2004 ) and PTDs (Belting, 2003 ). It has been suggested that GAG constitutes a cell-surface receptor for extracellular-peptide-carrier molecules that are associated or not with cargoes (Rusnati et al., 1999 , 2001 Belting, 2003 ). The initial step for several CPPs, including the arginine-rich Tat and pAnt peptides, is associated with strong electrostatic interactions with negatively charged GAGs, which trigger their internalization via different endocytosis pathways depending on the presence and the nature of the cargo and the ability of the CPP to interact with lipids (Suzuki et al., 2002 Console et al., 2003 Nakase et al., 2004 Wadia et al., 2004 Richard et al., 2005 ).
MPG carriers are amphipathic peptides bearing a hydrophobic domain derived from the fusion domain of HIV-1 gp41 (glycoprotein 41), and a NLS (nuclear localization sequence) with 5 positive charges. MPGs are able to form stable complexes with nucleic acids and improve their cellular uptake, and therefore their associated biological response. They have been largely used to improve the delivery of antisense oligonucleotides (Morris et al., 1997 ), plasmid DNA (Morris et al., 1999a ), siRNAs (small interferring RNAs) (Simeoni et al., 2003 Morris et al., 2004 Langlois et al., 2005 ) and peptides (Morris et al., 1999b ). Two MPG peptides have been developed called MPG-α and MPG-β which differ in their secondary structure when within the lipid membrane (Deshayes et al., 2004a , 2004b ). It has been reported that the uptake mechanism of biologically active MPG—cargo complexes is independent of the endosomal pathway and associated with the ability of MPG to interact with lipids and to induce local membrane destabilization (Deshayes et al., 2004a , 2004b ). Nevertheless, the parameters associated with the initiation of the cellular uptake mechanism of these CPPs are still poorly understood. In the present work, we have investigated the first step of the cellular uptake mechanism of both MPG-β and MPG-α. We have demonstrated that both MPG and MPG—cargo complexes interact with the negatively charged GAG of the extracellular matrix. The binding of MPG to GAG triggers specific activation of Rac1 GTPase, which is associated with the remodelling of the actin network, thereby constituting the ‘onset’ of cellular uptake and promoting the entry into the cell of MPG or MPG—DNA complexes and, for most CPPs, by increased membrane fluidity. Our results suggest that, although cell entry of CPPs can follow different pathways, there are some common initial steps which involve the GAG platform and GTPase activation.
Peptides and peptidomimetics as regulators of protein–protein interactions
Protein–protein interactions are essential for almost all cell processes.
Peptides are ideal candidates for targeting protein–protein interactions (PPIs).
We survey screening and rational design methods for identifying peptides to inhibit PPIs.
The increasing popularity of peptides as therapeutics may bring on a new era of drug discovery.
Protein–protein interactions are essential for almost all intracellular and extracellular biological processes. Regulation of protein–protein interactions is one strategy to regulate cell fate in a highly selective manner. Specifically, peptides are ideal candidates for inhibition of protein–protein interactions because they can mimic a protein surface to effectively compete for binding. Additionally, peptides are synthetically accessible and can be stabilized by chemical modifications. In this review, we survey screening and rational design methods for identifying peptides to inhibit protein–protein interactions, as well as methods for stabilizing peptides to effectively mimic protein surfaces. In addition, we discuss recent applications of peptides to regulate protein–protein interactions for both basic research and therapeutic purposes.
1 Cell-penetrating peptides: what are they?
The identification of proteins that can enter cells was first reported in the late eighties, contradicting the acknowledged understanding that the plasma membrane is impermeable to hydrophilic molecules. Thus, it has been demonstrated that the Trans-Activator of Transcription (Tat) protein of the Human Immunodeficiency Virus was able to efficiently enter tissue-cultured cells and promote the viral gene expression [1, 2] . Moreover, Antennapedia homeodomain, a transcription factor of Drosophilia melanogaster, was also shown to enter nerve cells and regulate neural morphogenesis  . The interesting spontaneous entry of both proteins led to extensive structure/function studies to find the shortest amino acid sequence necessary for the uptake. This resulted in the identification of the first CPPs: Tat peptide, corresponding to the basic domain of HIV-1 Tat protein [4, 5] and penetratin, corresponding to the third helix of the Antennapedia homeodomain  . Ever since, various peptides showing the same penetrating capacities have been discovered or rationally designed.
1.1 Definition and classification of CPPs
The field of CPPs evolved rapidly, ever since the first sequences were described. This makes it hard to have a general definition covering the characteristics of the different CPPs discovered. So far, one can say that CPPs are short peptides (generally not exceeding 30 residues) that have the capacity to ubiquitously cross cellular membranes with very limited toxicity, via energy-dependent and/or independent mechanisms, without the necessity of a chiral recognition by specific receptors. Most common CPPs are positively charged peptides, though the presence of few anionic or hydrophobic CPPs was also demonstrated. A primary or secondary amphipathic character is also implicated but not strictly required for the internalization.
According to their origin, we can distinguish three main classes of CPPs: peptides derived from proteins, chimeric peptides that are formed by the fusion of two natural sequences, and synthetic CPPs which are rationally designed sequences usually based on structure–activity studies (Table 1). Other attempts to classify CPPs, in spite of their diversity, were based on the physico-chemical characteristics of the sequences (e.g., their amphipathicity  , or their hydrophobicity  ). A recent review summarizes the different classifications and the physico-chemical properties of the so-far described CPPs  .
|Pep-1||HIV-reverse transcriptase/SV40 T-antigen||KETWWETWWTEWSQPKKKRKV|||
|Polyarginines||Based on Tat peptide||(R) n 6 < n < 12||[14, 15]|
|R6W3||Based on penetratin||RRWWRRWRR|||
CPPs can transport inside living cells a variety of covalently or non-covalently linked cargoes, as has been reviewed for nanoparticles  , peptides [18, 19] , proteins [20, 21] , antisense oligonucleotides [20, 22] , small interfering RNA  , double stranded DNA  and liposomes  .
The transport of the smallest cargo to large 120 kDa proteins had been successfully carried out both in vitro and in vivo. For instance, activable CPPs (ACPPs) were recently employed in vivo to target cancer cells over-expressing metalloproteinase-2  , while treatment of various inflammatory diseases by inhibition of NF-κB was also effective in vivo by coupling the inhibitors to different CPPs  . Tumor-targeting was also achieved in vivo for the (D)R8–doxorubicin conjugate  . It is difficult to keep track of the various applications because the field is emerging rapidly. Recently, a novel class of intrinsically bioactive CPPs, baptized bioportide, made an appearance with the description of a CPP sequence derived from cytochrome c that mimicked the apoptotic role of the entire protein once it entered inside cells [27, 28] . Another in vitro study on mouse neuronal hypothalamic cells revealed that the N-terminal sequence derived from the prion protein could penetrate cells and disabled the formation of prions  .
All cell lines were obtained from the American Type Culture Collection (ATCC) and passaged using standard techniques. The HeLa cell line was maintained and tested in Dulbecco’s modified Eagle medium (DMEM) high glucose, 10% fetal calf serum (FCS), and 1% antibiotic𠄺ntimycotic (ABAM) solution. The MDA-MB-231 cell line culture medium was Roswell Park Memorial Institute (RPMI) Medium 1640 (Gibco) supplemented with 9.5% FCS, 0.25 IU mL 𠄱 human insulin (Actrapid Penfill), and 20 mM HEPES (Gibco). The MCF-10A culture medium consisted of DMEM/F12 (Gibco) supplemented with 5% horse serum, human insulin (10 μg mL 𠄱 ), hydrocortisone (0.5 μg mL 𠄱 ), EGF (20 ng mL 𠄱 ), and cholera toxin (100 ng mL 𠄱 ).
All peptides were prepared, either commercially (Purar Chemicals, Australia) or in-house, by solid-phase peptide synthesis using Fmoc chemistry and Rink amide resin (except for FITC-labeled peptides, which utilized Fmoc-Val-Wang resin) and cleavage from the resin using standard procedures. The solution concentration of the peptides was determined spectrophotometrically at 280 nm using extinction coefficients determined from the amino acid content, 36 except for FITC-labeled peptides that were quantitated on the basis of the absorbance of fluorescein at 498 nm. 37
Biotinylated Penetratin Biotin-P16 (biotin-ahx-RQIKIWFQNRRMKWKK, where ahx = 1,6-aminohexanoic acid) was prepared with biotin linked N-terminally via the ahx group that served as a spacer. The peptide was purified to homogeneity using reversed phase high-performance liquid chromatography (RP-HPLC), and its identity was confirmed using mass spectrometry (calcd m/z (C120H194N38O22S2) 5+ : 517.7 found (C120H194N38O22S2) 5+ : 518.1).
Biotinylated N-terminal seven residues of Penetratin Biotin-P7 (biotin-βG-RRMKWKK, where βG = β-glycine) was prepared with biotin linked N-terminally via the β-glycine residue that served as a spacer. The peptide was purified to homogeneity using RP-HPLC, and its identity was confirmed using mass spectrometry (calcd m/z (C59H101N21O10S2) 4+ : 332.9 found (C59H101N21O10S2) 4+ : 333.0).
The control peptides Penetratin P16 (RQIKIWFQNRRMKWKK) and G7-18NATE (cyclo-CH2CO-WFEGYDNTFPC) were prepared as reported previously. 35,38
Cargo-containing peptides G7-18NATE-P16 (cyclo-(CH2CO-WFEGYDNTFPC-RQIKIWFQNRRMKWKK)) and G7-18NATE-P7 (cyclo-(CH2CO-WFEGYDNTFPC-RRMKWKK)) were synthesized as a continuous peptide chain and then cyclized via the formation of thioether between the N-terminus and the cysteine side chain post cleavage, as described for G7-18NATE. 38 The peptides were purified to homogeneity using RP-HPLC, and their identities were confirmed using mass spectrometry G7-18NATE-P16 (calcd m/z (C171H246N48O38S2) 6+ : 608.3 found (C171H246N48O38S2) 6+ : 608.6) and G7-18NATE-P7 (calcd m/z (C113H159N31O26S2) 5+ : 487.03 found (C113H159N31O26S2) 5+ : 487.25).
FITC-labeled peptides FITC-P16-NLS (FITC-ahx-RQIKIWFQNRRMKWKK-PKKKRKV), FITC-P7-NLS (FITC-ahx-RRMKWKK-PKKKRKV), and FITC-NLS (FITC-ahx-PKKKRKV) were prepared with FITC linked N-terminally via the ahx group (1,6-aminohexanoic acid), which served as a spacer. The peptides were purified to homogeneity using RP-HPLC, and their identities were confirmed using mass spectrometry FITC-P16-NLS (calcd m/z (C171H266N50O33S2) 6+ : 603.0 found (C171H266N50O33S2) 6+ : 603.46), FITC-P7-NLS (calcd m/z (C113H179N33O21S2) 4+ : 600.59 found (C113H179N33O21S2) 4+ : 601.03), and FITC-NLS (calcd m/z (C67H100N16O14S) 3+ : 462.58 found (C67H100N16O14S) 3+ : 462.72).
Peptide Internalization Assay
HeLa cells were seeded in 96-well plates (30 cells per well) in a culture medium. After 24 h, the media was replaced with a media containing the peptide of interest (at 1 μM) and incubated for 15 min. The cells were washed three times in ice-cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde and permeabilized with 0.5% Tween20/PBS. The endogenous alkaline phosphatase activity was neutralized by incubating the plates at 65 ଌ for 60 min. The cells were blocked using 3% bovine serum albumin (BSA)/PBS and treated with streptavidin APase (1 μg mL 𠄱 in 0.1% BSA/PBS) for 30 min at room temperature. Following three times washing with PBS, 50 μL of 100 mg.mL 𠄱 p-nitrophenol phosphate was introduced and incubated for 30 min at room temperature. The enzyme reaction was quenched using 2 M NaOH. The absorbance was measured at 405 nm using FLUOstar Omega. The samples were measured in triplicates, and the background absorbance was determined using untreated cells and subtracted from the test conditions. The statistical analysis was performed using GraphPad Prism6 (GraphPad Software, CA).
Wound-Closure Migration Assay
The MDA-MB-231 or MCF-10A cells were seeded until confluent in 24-well plates, wounded with a sterile pipette tip to create a cell-free gap and cell debris cleared away with fresh media. Lyophilized peptides were resuspended in sterile MQ, diluted to the appropriate concentration in fresh media, and added to the appropriate wells, with each treatment duplicated. The media also contained 1 ng mL 𠄱 EGF to stimulate migration. The Leica AF6000 LX live-cell-imaging system was used to capture images in real time (at 37 ଌ, 5% CO2), with images collected every 30 min from a minimum of five positions per well. The remaining gap area was measured using ImageJ (Fiji) at 0 and 12 h and averaged from all measured positions across the duplicates. At least three independent experiments were performed. To allow for a direct comparison, the results are displayed as the relative percentage of gap closure compared with the untreated control cells normalized to 100%.
Cellular Uptake Assay
MDA-MB-231 cells were seeded (50) in 24-well plates and grown until 60% confluent. The media was replaced with the peptide-treated media (at 20 μM) and incubated for 20 min at 37 ଌ. The cells were washed gently three times with the growth media and incubated with Hoechst-treated media (1 μM) for 5 min at 37 ଌ before being transferred to the Leica AF6000 LX live-cell-imaging system. Here, the cells were maintained at 37 ଌ and 5% CO2, and images were taken within 15 min using a 20× objective lens.
Expression and Purification of the Grb7-SH2 Domain
Grb7-SH2 (415 residues) was incorporated into the pGex2T plasmid and expressed and purified, as described previously. 39
Thermal Shift Assays
The lyophilized peptides of interest were resuspended in 50 mM NaPO4, 150 mM NaCl, 1 mM dithiothreitol, and 5% (v/v) dimethyl sulfoxide (DMSO) and tested at a final concentration of 50 μM. Grb7-SH2, dialyzed in the same buffer (excluding DMSO), was tested at 40 μM. Using Rotor-Gene 3000 (Corbett Life Science), the temperature was increased in 0.5 ଌ increments from 50 to 70 ଌ with a holding time of 60 s. The wavelength of excitation/emission was measured at 530/555 nm. Each condition was measured in duplicate or triplicate with values averaged. Peptide-only and buffer-only control samples were measured and subtracted from the averaged test measurements. At least three independent experiments were conducted. The statistical analysis was performed using GraphPad Prism6 (GraphPad Software, CA).