Targeting heat shock proteins on cancer cells: selection, characterization, and cell-penetrating properties of a peptidic GRP78 ligand

Peptidic ligands can be used for specific cell targeting and the delivery of payloads into the target cell. Here we describe the screening of a pool of cyclic peptide phage display libraries using whole-cell panning against human melanoma cell line Me6652/4. This strategy resulted in the selection of the cyclic 13-mer Pep42, CTVALPGGYVRVC, which showed preferential internalization into melanoma cell line Me6652/4 versus the reference cell line Me6652/56. This translocation is a receptor-mediated process that does not require electrostatic interactions nor does it involve transfer to the lysosomal compartment. The cellular receptor for Pep42 was identified as the surface membrane form of glucose-regulated protein 78 (GRP78), a member of the heat shock protein family and a marker on malignant cancer cells. The cellular uptake and intracellular trafficking of Pep42-Quantum Dot conjugates was monitored by confocal laser microscopy, and colocalization within the endoplasmic reticulum was observed. The uptake of Pep42 could be blocked by a monoclonal antibody against the identified receptor. Furthermore, Pep42 was shown to target specifically GRP78-expressing cancer cells. The in vitro cytotoxicity of a Pep42-Taxol conjugate was evaluated by flow cytometry wherein the conjugate was shown to induce apoptosis and was more effective in promoting programmed cell death in Me6652/4 cells. In summary, the data presented suggest that cyclic peptide Pep42 might be a powerful tool in the construction of drug conjugates designed to selectively kill malignant cancer cells.     

Synthesis,characterization, and biological activity of poly(arginine)‐derived cancer‐targeting peptides in HepG2 liver cancer cells

The solid‐phase synthesis, structural characterization, and biological evaluation of a small library of cancer‐targeting peptides have been determined in HepG2 hepatoblastoma cells. These peptides are based on the highly specific Pep42 motif, which has been shown to target the glucose‐regulated protein 78 receptors overexpressed and exclusively localized on the cell surface of tumors. In this study, Pep42 was designed to contain varying lengths (3–12) of poly(arginine) sequences to assess their influence on peptide structure and biology. Peptides were effectively synthesized by 9‐fluorenylmethoxycarbonyl‐based solid‐phase peptide synthesis, in which the use of a poly(ethylene glycol) resin provided good yields (14–46%) and crude purities >95% as analyzed by liquid chromatography–mass spectrometry. Peptide structure and biophysical properties were investigated using circular dichroism spectroscopy. Interestingly, peptides displayed secondary structures that were contingent on solvent and length of the poly(arginine) sequences. Peptides exhibited helical and turn conformations, while retaining significant thermal stability. Structure–activity relationship studies conducted by flow cytometry and confocal microscopy revealed that the poly(arginine) derived Pep42 sequences maintained glucose‐regulated protein 78 binding on HepG2 cells while exhibiting cell translocation activity that was contingent on the length of the poly(arginine) strand. In single dose (0.15 mM) and dose‐response (0–1.5 mM) cell viability assays, peptides were found to be nontoxic in human HepG2 liver cancer cells, illustrating their potential as safe cancer‐targeting delivery agents. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Functional and structural characteristics of anticancer peptide Pep27 analogues

Background  

A secreted peptide Pep27 initiates the cell death program in S. pneumoniae through signal transduction. This study was undertaken to evaluate the relation between the structure and cytotoxic activity of Pep27 and its analogues on cancer cells.     

A lactoferrin-derived peptide with cationic residues concentrated in a region of its helical structure induces necrotic cell death in a leukemic cell line (HL-60).

Model studies have shown that peptides derived from the N-terminal region of bovine lactoferrin (Lf-B) exhibit antitumor activity against certain cell lines. This activity is due primarily to the peptides' apoptogenic effect. Several reports indicate that cationic residues clustered in two regions of the peptide sequence can be shuffled into one region and thereby increase cytotoxic activity, although the mechanism of this enhanced cytotoxic effect has not been clarified. In this paper, we considered several parameters that determine the mode of cell death after exposure to a native Lf-B derived peptide (Pep1, residues 17-34), and a modified peptide (mPep1) wherein the cationic residues of Pep1 are clustered in a single region of its helical structure. We found that the cytotoxic activity of mPep1 was about 9.6 fold-higher than that of Pep1 against HL-60 cells, as determined by the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) assay. In investigating the expression of phosphatidylserine, we observed that the native peptide (Pep1) caused both apoptotic cell death and necrotic cell death, depending on the concentration of the peptide. In contrast, the action of mPep1 was exclusively characteristic of necrotic cell death. This observation was further confirmed by agarose gel electrophoresis, in which clear ladder-like DNA bands were observed from cells exposed to Pep1, whereas DNA from cells treated with mPep1 produced a smeared pattern. We extended the study by investigating the release of mitochondrial cytochrome c into the cytosol, and the activation of caspase-3; both peptides caused the release of cytochrome c into the cytosol, and the activation of caspase-3.These results suggest that Pep1 may kill cancer cells by activating an apoptosis-inducing pathway, whereas mPep1 causes necrotic cell death by destroying cellular membrane structure notwithstanding sharing some cellular events with apoptotic cell death. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Genetic Mapping of Genes Concerned with Glutamyl Polypeptide Production by Bacillus licheniformis and a Study of Their Relationship to the Development of Competence for Transformation

By transduction with SP-15, we have mapped some of the genetic sites involved in glutamyl polypeptide (Pep) production by Bacillus licheniformis 9945A. Eleven peptide mutations were assigned to group 1 which lies between the gly-42 and his-6 markers. Three other mutations were assigned to group 2 which lies between the ade-2 and str-1 sites. A series of 28 additional Pep(-) mutants did not belong to either group 1 or 2; the chromosomal location of those peptide mutations is unknown. All three mutants of group 2 were highly transformable, but only two of group 1 transformed well under the conditions employed. Knowing the chromosomal markers that were linked to peptide mutations made it possible to investigate the effect peptide mutations might have on the development of competence for transformation. Nontransformable organisms were rendered transformable upon the introduction, by transduction, of Pep(-) mutations from transformable strains. These results supported the conclusion that the ability of cells to develop competence for transformation when grown under appropriate conditions was related to the physiological effects of particular peptide mutations.     

Computational modeling of novel inhibitory peptides targeting proteoglycan like region of carbonic anhydrase IX and in vitro validation in HeLa cells

Abstract

Carbonic anhydrase IX (CAIX) is a tumour-associated, hypoxia-induced, membrane-bound metallo-enzyme which catalyzes the reversible hydration of carbon dioxide (CO₂) to bicarbonate (HCO₃^?) and proton (H⁺) ions. Over expression of CAIX is observed in cancers of colon, lung, kidney, breast, etc. CAIX plays a vital role in maintaining favourable intracellular pH for tumour cell growth and extracellular acidification which in-turn leads to drug resistance and spread of factors influencing tumour invasion. The N-terminal proteoglycan (PG) – like fragment of CAIX is unique to this isoform and is considered as potential druggable hotspot. Recently, M75 monoclonal antibody targeting the LPGEEDLPG epitope of PG like region has been proposed to reduce cellular adhesion in cancer cells. LPGEEDLPG fragment in complex with M75 has been crystallized and it serves as a strong base for development of peptide inhibitors based on interacting interfaces. Thus, in this study, an in-depth analysis of intermolecular interactions in LPGEEDLPG-M75 complex was carried out by implementing extensive molecular dynamics simulations, binding free energy calculations so as to infer the major determinant fragments of M75 that can be used as peptide inhibitors targeting PG region. Based on these analyses, 3 peptides (Pep1, Pep2 and Pep3) were synthesized and validated by in vitro assays involving cytotoxicity assessment, CAIX inhibition analysis through Direct and Indirect functional assays, and inhibition of Cell adhesion in HeLa cells. The results reveal Pep1 to be a promising inhibitor as it could efficiently modulate CAIX mediated pH homeostasis and cell adhesion in cancer cells.

Communicated by Ramaswamy H. Sarma     

Influence of the staphylococcinlike peptide Pep 5 on membrane potential of bacterial cells and cytoplasmic membrane vesicles.

The staphylococcinlike peptide Pep 5 rapidly abolished the membrane potential of bacterial cells; active transport of amino acids by cytoplasmic membrane vesicles was inhibited and preaccumulated amino acids were released upon the addition of Pep 5. Artificial asolectin vesicles were not impaired by the peptide. It is concluded that the cytoplasmic membrane is the primary target of Pep 5.     

Haemolysin-derived synthetic peptides with pore-forming and haemolytic activity

Escherichia coli haemolysin (Hlya) is a pore-forming protein which belongs to the family of 'Repeat-toxins' (RTX) (Lo et al., 1987; Lally et al., 1989; Kraig et al., 1990). A model for the pore-forming structure of HlyA has been proposed (Ludwig et al., 1991) which consists of eight transmembrane segments all present in this hydrophobic region of HlyA. We report here that two synthetic peptides of 10 and 8 amino acids in length (Pep1 and Pep2, respectively), which are derived from transmembrane segment V, are able to form pores in an artificial lipid bilayer. In addition, Pep1 exhibits strong haemolytic activity when tested on human red blood cells (HRBCs). The haemolytic activity of Pep1 and of E. coli haemolysin is completely inhibited by antibodies raised against Pep1.     

Mitochondrial degradation in acetic acid‐induced yeast apoptosis: the role of Pep4 and the ADP/ATP carrier

We have previously shown that acetic acid activates a mitochondria‐dependent death process in Saccharomyces cerevisiae and that the ADP/ATP carrier (AAC) is required for mitochondrial outer membrane permeabilization and cytochrome c release. Mitochondrial fragmentation and degradation have also been shown in response to this death stimulus. Herein, we show that autophagy is not active in cells undergoing acetic acid‐induced apoptosis and is therefore not responsible for mitochondrial degradation. Furthermore, we found that the vacuolar protease Pep4p and the AAC proteins have a role in mitochondrial degradation using yeast genetic approaches. Depletion and overexpression of Pep4p, an orthologue of human cathepsin D, delays and enhances mitochondrial degradation respectively. Moreover, Pep4p is released from the vacuole into the cytosol in response to acetic acid treatment. AAC‐deleted cells also show a decrease in mitochondrial degradation in response to acetic acid and are not defective in Pep4p release. Therefore, AAC proteins seem to affect mitochondrial degradation at a step subsequent to Pep4p release, possibly triggering degradation through their involvement in mitochondrial permeabilization. The finding that both mitochondrial AAC proteins and the vacuolar Pep4p interfere with mitochondrial degradation suggests a complex regulation and interplay between mitochondria and the vacuole in yeast programmed cell death.     

Susceptibility of bacterial, eukaryotic and artificial membranes to the disruptive action of the cationic peptides Pep 5 and nisin

Abstract The cationic bactericidal peptides Pep 5 and nisin render membranes permeable to low- M _r compounds. All Gram-positive bacteria treated with these peptides showed an immediate efflux of entrapped radioactive markers. The uptake of α-[¹⁴C]methylglucoside by the phosphoenolpyruvate-dependent phosphotransferase system was stimulated by Pep 5, supporting previous results that pep 5 abolishes the membrane potential. Oxygen consumption was inhibited, presumably due to lack of ADP. Escherichia coli became sensitive to Pep 5 and nisin when the outer membrane was bypassed by osmotic shock or by formation of cytoplasmic membrane vesicles. In contrast, Mycoplasma cells and erythrocytes were unaffected by Pep 5 and nisin in concentrations up to 1 mM. Human lung fibroblasts released only small amounts of ATP when treated with Pep 5 and nisin in μM concentrations. Eukaryotic and Mycoplasma cells were disrupted more effectively by the bee venom peptide melittin, which displays overall structural similarities to Pep 5 and nisin. Various artificial membranes were not affected by Pep 5, nisin, or melittin.     

Autographa californica multiple nucleopolyhedrovirus nucleocapsid protein BV/ODV-C42 mediates the nuclear entry of P78/83

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) BV/ODV-c42 (orf101; c42), which encodes a 41.5-kDa viral nucleocapsid protein with a putative nuclear localization signal (NLS) motif at the C terminus, is a highly conserved gene among members of the Baculoviridae family. C42 is demonstrated to be essential for AcMNPV propagation and can bind to nucleocapsid protein P78/83, a viral activator for the actin-related protein 2/3 (ARP2/3) complex to initiate nuclear actin polymerization, which is essential for viral nucleocapsid morphogenesis during AcMNPV infection. Here, we report the identification of a novel pathway through which c42 functions in nucleocapsid assembly. Cotransfection of Sf9 cells with c42 and p78/83 plasmids demonstrated that C42 was capable of recruiting P78/83 to the nuclei of uninfected cells and that the NLS motif of C42 was essential for this process. To validate this nuclear relocation mode in bacmid-transfected cells, a c42-disrupted bacmid (vAc(c42ko-gfp)) and rescued bacmids with wild-type c42 (vAc(c42res-gfp)) or with NLS coding sequence-mutated c42 (vAc(c42nls-gfp)) were prepared. By immuno-staining, P78/83 was found to be localized in the cytoplasm of either vAc(c42ko-gfp)- or vAc(c42nls-gfp)-transfected cells, whereas P78/83 was relocated to the nuclei of vAc(c42res-gfp)-transfected cells. Furthermore, F-actin-specific staining confirmed that there was no actin polymerization activity in the nuclei of either vAc(c42ko-gfp)- or vAc(c42nls-gfp)-transfected cells, which might be attributed to the absence of nuclear P78/83, an activator of the ARP2/3 complex to initiate nuclear actin polymerization. We therefore hypothesize a mode of action where C42 binds to P78/83 in the cytoplasm to form a protein complex and cotransports to the nucleus under the direction of the NLS motif in C42 during AcMNPV infection.     

Induction of autolysis of staphylococci by the basic peptide antibiotics Pep 5 and nisin and their influence on the activity of autolytic enzymes

Pep 5 and nisin are cationic bactericidal peptides which were shown to induce autolysis in Staphylococcus cohnii 22. In contrast to nisin, Pep 5 induced lysis could be stimulated in the presence of glucose. Addition of lipoteichoic acids (LTA) (d-alanine:phosphorus=0.475:1) inhibited all effects of Pep 5 on susceptible cells in a molar ratio LTA:Pep 5 of 10:1. Treatment of S. cohnii 22 with Pep 5 or nisin for 20 min and subsequent washing with 2.5 M NaCl released autolysin activity. Crude preparations of the hydrolyzing enzymes produced free amino groups as well as polysaccharide fragments from the murein backbone, suggesting the presence of a muramidase or glucosamidase, and endopeptidase or amidase. Both enzyme activities were inhibited by lipoteichoic acid; they could be fully reactivated by addition of Pep 5 in sufficient concentrations. The velocity of hydrolysis was not influenced by nisin, whereas it was doubled in presence of Pep 5. The results are discussed in view of a possible mechanism of induction of lysis by Pep 5 and nisin.Abbreviations A.U. arbitrary unit - CCCP carbonylcyanide-m-chlorophenyl hydrazone - DNase deoxyribonuclease - CYG casein yeast extract glucose - IT initial turbidity - LTA lipoteichoic acid - RNase ribonuclease - TSB Tryptone Soy Broth     

A selective transport route from Golgi to late endosomes that requires the yeast GGA proteins

Pep12p is a yeast syntaxin located primarily in late endosomes. Using mutagenesis of a green fluorescent protein chimera we have identified a sorting signal FSDSPEF, which is required for transport of Pep12p from the exocytic pathway to late endosomes, from which it can, when overexpressed, reach the vacuole. When this signal is mutated, Pep12p instead passes to early endosomes, a step that is determined by its transmembrane domain. Surprisingly, Pep12p is then specifically retained in early endosomes and does not go on to late endosomes.By testing appropriate chimeras in mutant strains, we found that FSDSPEF-dependent sorting was abolished in strains lacking Gga1p and Gga2p, Golgi-associated coat proteins with homology to gamma adaptin. In the gga1 gga2 double mutant endogenous Pep12p cofractionated with the early endosome marker Tlg1p, and recycling of Snc1p through early endosomes was defective. Pep12p sorting was also defective in cells lacking the clathrin heavy or light chain. We suggest that specific and direct delivery of proteins to early and late endosomes is required to maintain the functional heterogeneity of the endocytic pathway and that the GGA proteins, probably in association with clathrin, help create vesicles destined for late endosomes.     

Pep3p/Pep5p complex: a putative docking factor at multiple steps of vesicular transport to the vacuole of Saccharomyces cerevisiae

Pep3p and Pep5p are known to be necessary for trafficking of hydrolase precursors to the vacuole and for vacuolar biogenesis. These proteins are present in a hetero-oligomeric complex that mediates transport at the vacuolar membrane. PEP5 interacts genetically with VPS8, implicating Pep5p in the earlier Golgi to endosome step and/or in recycling from the endosome to the Golgi. To understand further the cellular roles of Pep3p and Pep5p, we isolated and characterized a set of pep3 conditional mutants. Characterization of mutants revealed that pep3(ts) mutants are defective in the endosomal and nonendosomal Golgi to vacuole transport pathways, in the cytoplasm to vacuole targeting pathway, in recycling from the endosome back to the late Golgi, and in endocytosis. PEP3 interacts genetically with two members of the endosomal SNARE complex, PEP12 (t-SNARE) and PEP7 (homologue of mammalian EEA1); Pep3p and Pep5p associate physically with Pep7p as revealed by two-hybrid analysis. Our results suggest that a core Pep3p/Pep5p complex promotes vesicular docking/fusion reactions in conjunction with SNARE proteins at multiple steps in transport routes to the vacuole. We propose that this complex may be responsible for tethering transport vesicles on target membranes.     

Peptide-like substances as antimicrobial barriers to Corynebacterium sp. adhesion to silicone catheters

AIMS: To show medical application of antimicrobial peptides such as Pep5 and epidermin in inhibiting adhesion of Corynebacterium spp. to silicone catheters. METHODS AND RESULTS: The inhibitory activity of crude preparations of Pep5 and epidermin was tested on Corynebacterium spp. isolated from catheter-related infections. The addition of these substances at 640 AU ml(-1) to a cell suspension of Corynebacterium sp. 633544 resulted in a decrease of 3 log cycles in the number of viable cells over a period of 12 h. When Pep5 and epidermin were added to in vitro catheter colonization experiments, there was a decrease of 1 log unit (P < 0.01) in the cell number of Corynebacterium spp. adhered to silicone catheters. Scanning electron microscopy revealed that antimicrobial-treated catheters presented zones with absence of adhered cells, and some parts of the catheter presented aggregates suggesting damaged cells. CONCLUSIONS: The crude preparations of Pep5 and epidermin were able to inhibit Corynebacterium sp. 633544 isolated from catheter-related infection. The capability of Pep5 and epidermin to inhibit catheter colonization may indicate their usefulness as a barrier to block or to reduce the bacteremia by Corynebacterium spp. SIGNIFICANCE AND IMPACT OF THE STUDY: Peptide-like antimicrobial substances used to reduce bacterial attachment to medical devices may represent a novel strategy to control catheter-related infections.     

Structural analysis of immunotherapeutic peptides for autoimmune Myasthenia gravis

Myasthenia gravis (MG) and its animal model, experimental MG (EAMG), are autoimmune disorders in which major pathogenic antibodies are directed against the main immunogenic region (MIR) of the nicotinic acetylcholine receptor (nAChR). In an earlier attempt to develop peptide mimotopes capable of preventing the anti-MIR-mediated pathogenicity, the peptide Pep.1 was initially identified from phage display, and subsequently, Cyclic extended Pep.1 (Cyc.ext.Pep.1), which incorporates eight additional residues into the Pep.1 sequence and has an affinity for the anti-MIR antibody mAb198 3 orders of magnitude greater than that of Pep.1, was developed. In an animal model, Pep.1 shows no ability to inhibit mAb198-induced EAMG, whereas Cyc.ext.Pep.1 successfully blocks anti-MIR antibody 198 (mAb198)-induced EAMG. Our aim in this study was to identify the structural characteristics related to the different affinities for mAb198 of Pep.1 and Cyc.ext.Pep.1 using NMR spectroscopy and alanine scanning analysis. The NMR structural analysis revealed that Pep.1 is very flexible in solution, whereas Cyc.ext.Pep.1 is highly rigid within a region containing several turn structures. Interestingly, TRNOE experiments revealed that mAb198-bound Pep.1, particularly in the region between Asn7 and Glu11, shows significant structural similarity to the region between Asn10 and Glu14 of Cyc.ext.Pep.1, which is critical for interaction with mAb198. We therefore conclude the higher affinity of Cyc.ext.Pep.1 for mAb198 reflects the fact that incorporation of additional residues producing a single disulfide bond endows Pep.1 with a conformational rigidity that mimics the structure of mAb198-bound Pep.1. Furthermore, our results suggest that cyclic extended peptides could be utilized generally as useful tools to optimize the affinity of phage library-derived peptide antigens.     

Mode of action of the staphylococcinlike peptide Pep 5: voltage-dependent depolarization of bacterial and artificial membranes.

The cationic staphylococcinlike peptide Pep 5 is shown to depolarize bacterial and planar lipid membranes in a voltage-dependent manner. An artificial valinomycin-induced potassium diffusion potential across the cytoplasmic membrane of Staphylococcus cohnii 22 was sufficient to promote Pep 5 action. Thus, evidence is provided that a membrane potential of sufficient magnitude is the only prerequisite for Pep 5 activity. The voltage dependence was elucidated by macroscopic conductance measurements with black lipid membranes. A threshold potential of about -90 to -100 mV, which was deduced from experiments with bacterial cells, could be confirmed. Single pores were resolved which often occur as short-lived bursts and fluctuate among different conductance levels. Pore diameters were calculated ranging from 0.1 to 1 nm. Succinylation of the lysine residues of Pep 5 resulted in prolonged pore lifetimes and maintenance of distinct conductance levels. However, the succinylated peptide required a higher threshold potential, approximately -150 mV, than the native peptide, which is probably the reason for the reduced activity of the modified peptide against intact gram-positive bacteria.     

Inhibition of plant-pathogenic bacteria by short synthetic cecropin A-melittin hybrid peptides

Short peptides of 11 residues were synthesized and tested against the economically important plant pathogenic bacteria Erwinia amylovora, Pseudomonas syringae, and Xanthomonas vesicatoria and compared to the previously described peptide Pep3 (WKLFKKILKVL-NH(2)). The antimicrobial activity of Pep3 and 22 analogues was evaluated in terms of the MIC and the 50% effective dose (ED(50)) for growth. Peptide cytotoxicity against human red blood cells and peptide stability toward protease degradation were also determined. Pep3 and several analogues inhibited growth of the three pathogens and had a bactericidal effect at low micromolar concentrations (ED(50) of 1.3 to 7.3 microM). One of the analogues consisting of a replacement of both Trp and Val with Lys and Phe, respectively, resulted in a peptide with improved bactericidal activity and minimized cytotoxicity and susceptibility to protease degradation compared to Pep3. The best analogues can be considered as potential lead compounds for the development of new antimicrobial agents for use in plant protection either as components of pesticides or expressed in transgenic plants.     

The Syntaxin Tlg1p Mediates Trafficking of Chitin Synthase III to Polarized Growth Sites in Yeast

Tlg1p and Tlg2p, members of the syntaxin family of SNAREs in yeast, have been implicated in both endocytosis and the retention of late Golgi markers. We have investigated the functions of these and the other endocytic syntaxins Pep12p and Vam3p. Remarkably, growth is possible in the absence of all four proteins. In the absence of the others, Pep12p and Tlg1p can each create endosomes accessible to the endocytic tracer dye FM4-64. However, although Pep12p is required for the ligand-induced internalization of the α factor receptor and its passage via Pep12p-containing membranes to the vacuole, Tlg1p is not. In contrast, Tlg1p is required for the efficient localization of the catalytic subunit of chitin synthase III (Chs3p) to the bud neck, a process that involves endocytosis and polarized delivery of Chs3p. In wild-type cells, internalized Chs3p cofractionates with Tlg1p and Tlg2p, and in a strain lacking the other endocytic syntaxins, either Tlg1p or Tlg2p is sufficient for correct localization of the enzyme. Pep12p is neither necessary nor sufficient for this process. We conclude that there are two endocytic routes in yeast that can operate independently and that Tlg1p is located at the junction of one of these with the polarized exocytic pathway.