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Hydrophobic Membrane Translocating Sequence Peptides 119 6.3.2 APPLICATIONS OF SN50 PEPTIDE The peptide SN50 is now used as a general reagent in molecular biology due to its proven ability to prevent NF-κB-induced gene expression. Kilgore et al. 41 used SN50 in a study of the role of NF-κB in the up-regulation of certain proteins during formation of the membrane attack complex (MAC) of complement. Following activation of the complement cascade, the assembly of the MAC induces production of inflammatory mediators, e.g., IL-8, monocyte chemoattractant protein-1 (MCP-1); however, the mechanism by which this occurred was unknown. Through a detailed assessment of the nuclear activity of NF-κB in the presence and absence of SN50 following MAC formation, the authors concluded that MAC formation may serve to activate IL-8 and MCP-1 genes through activation and translocation of NF-κB. In a second example of SN50 used as a general reagent, the peptide was employed to determine the role of NF-κB in the response of myocardial tissue to ischemia. Maulik et al. 42 speculated that NF-κB was involved in myocardial adaptation to ischemia. In this study rat hearts were rapidly excised and retrograde perfusion established before subjecting the hearts to ischemic stress and monitoring the adaptation. To define the role of NF-κB, the SN50 peptide was added to the perfusion buffer and hearts were exposed to 18 µM of the peptide over a 15-min period. It was found that the beneficial effects of ischemic adaptation were blocked by pretreating the hearts with SN50. This shows that SN50 is capable of translocating not only into cell suspensions but also into tissue. More examples of the recent uses of SN50 to delineate the role of NF-κB in signaling pathways are listed in Table 6.1. Results presented in these studies vary; in some cases NF-κB was found to be important in the pathway examined, but in other situations it has been determined that NF-κB is not involved. 6.3.3 KFGF MTS AIDS IN TRANSLOCATION OF OTHER CARGOES Based on early success of the delivery of NF-κB NLS into intact cells when covalently linked to the kFGF MTS, Lin et al. 34 proceeded to link the same MTS to a different NLS, this time derived from the protein fibroblast growth factor-1 (FGF-1). The purpose of this assembly was to produce a reagent allowing examination of the functional role of the NLS in FGF-1-stimulated DNA synthesis and mitogenesis. A stepwise solid-phase peptide synthesis method was employed to produce this chimeric peptide (SA), with the MTS located at the N terminus separated from the NLS by a three-residue spacer (AAA). An indirect immunofluorescence assay was used to show that SA peptide migrated into NIH 3T3 cells with localization to the perinuclear areas and a thymidine incorporation assay showed that the peptide induced a mitogenic response in a manner similar to the entire FGF-1 protein. A peptide representing the NLS alone was not capable of inducing such a response and it was therefore concluded that the entire SA peptide was required for cell penetration and biological activity. This same peptide was also found to be effective in a range of other cell types, i.e., BALB/c3T3 murine fibroblasts, human vascular endothelial cells, and primary cultured hepatocytes. 74
120 Cell-Penetrating Peptides: Processes and Applications TABLE 6.1 Recent Applications of SN50 to Investigate Role of NF-κB in Various Signaling Pathways Disease Function under investigation Ref. Cancer Viral infection Lyme disease Multiple sclerosis Huntington’s disease Parkinson’s disease Adult respiratory distress syndrome Leukemic cell proliferation via TNFα LHRH expression by ovarian cancers HeLa cell resistance to cisplatin Tumor cell adhesion via β1 integrin NGF as mitogen for breast cancer TRAIL as multiple myeloma therapeutic Protein regulation by cytomegalovirus HIV-1 Tat modulaiton of ion channels HIV-1 Tat modulation of cAMP Flavivirus-induced apoptosis and replication Upregulation of MM-9 by Borrelia burgdorferi Induction of iNOS in microglial cells c-Myc and p53 induction of apoptosis Dopamine-induced apoptosis Asbestos-induced TNFα gene expression Silica-induced TNFα−gene expression TNFα induction of chemokine IL-8 Asthma Role of GATA3 in Th2 differentiation Other Induction of chemokines by spider venom Expression of LIF and IL-6 in endometrium Cyanide-induced apoptosis TNFα regulation of corneal fibroblast apoptosis TNFα regulation of uterine cell death Generation of GM-CSF by human monocytes TNFα-induced apoptosis Induction of IL-6 by TNFα Survival of cerebellar granule neurons Induction of cytokines by macrophages Insulin rescue of retinal neurons 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67–69 70 71 72 73 The kFGF MTS has been covalently linked to several other peptides, including a cyclic, 10-residue inhibitor of enzyme oligosaccharyl transferase for translocation across the endoplasmic reticulum membrane in vitro; 75 a peptide representing an epidermal growth factor receptor (EGFR) autophosphorylation site; 35 a 12-residue peptide derived from the region surrounding phosphotyrosine 317 of the intracellular signaling protein Shc; 76 and a series of peptides representing binding sites of proteins involved in signaling pathways associated with activation of the G protein-coupled receptor 5-HT 2C in choroid plexus endothelial cells. 77 Moreover, the nuclear import of NF-κB has been inhibited by an SN50 analog, a peptide comprising two NLSs of NF-κB, one on either end of the kFGF MTS, to increase the efficacy of nuclear uptake. 78 This peptide was also synthesized using D-amino acids to increase resistance
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CELL- PENETRATING PEPTIDES Processe
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Pharmacology and Toxicology: Basic
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Library of Congress Cataloging-in-P
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to the handbook are prominent resea
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REFERENCES 1. Green, M. and Loewens
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Contributors Mats Andersson Microbi
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Erin T. Pelkey Department of Chemis
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Contents Section I Classes of Cell-
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Chapter 16 Cell-Penetrating Peptide
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The Tat-Derived Cell-Penetrating Pe
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24 Cell-Penetrating Peptides: Proce
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3 Transportans Margus Pooga, Mattia
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Transportans 55 Indeed, galparan is
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Transportans 57 especially the endo
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Transportans 59 In the penetration
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Transportans 61 A 21-mer antisense
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Transportans 63 Commonly, the prote
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Transportans 65 antibiotin antibodi
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Transportans 67 For cross-linking o
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Interactions of Cell-Penetrating Pe
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Structure Prediction of CPPs and It
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FIGURE 9.7 (Color Figure 9.7 follow
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FIGURE 9.8 The center of the figure
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Biophysical Studies of Cell-Penetra
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Toxicity and Side Effects of Cell-P
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264 Cell-Penetrating Peptides: Proc
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Kinetics of Uptake of Cell-Penetrat
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Cell-Penetrating Peptide Conjugatio
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Cell-Penetrating Peptides as Vector
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TABLE 16.1 Examples of Transport of
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CPP 2 Cargo or mRNA CAP Antisense A
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Microbial Membrane-Permeating Pepti
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Index A Abaecin, 129 Abz radiolabel
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Index 399 Diffraction, 168 Disulphi
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Index 401 structure prediction, 187
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Index 403 pRB proteins, Tat-E1A bin
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Index 405 lipid perturbation (secon
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