Cell type dependent endocytic internalization of ErbB2 with an artificial peptide ligand that binds to ErbB2

ErbB2, which is a member of the epidermal growth factor (erbB) receptor family, is frequently overexpressed in breast and ovarian cancers. Antibody and small molecule anti-tyrosine kinase inhibitors have been developed for targeted therapies for cancers overexpressing erbB2. Internalization and downregulation of erbB2, which is induced by a ligand, may be important for efficacious therapeutic effects. However, ligand-dependent erbB2 internalization has not been well characterized. Here we investigated the internalization of erbB2 in SKBr3 and SKOv3 cells, both overexpressing erbB2, using an EC-1 peptide fused to eGFP (EC-eGFP), which specifically binds to erbB2. ErbB2 was internalized in SKOv3 cells when the cells were treated with EC-eGFP. The accumulation of endosomal erbB2 was EC-eGFP dependent, which colocalized with transferrin implying endocytosis via clathrin-coated pits. In contrast, internalization of erbB2 was not observed in SKBr3 cells. As a result, two different mechanisms, which are cell type dependent for the internalization of erbB2, are proposed.     

Enhanced internalization of ErbB2 in SK-BR-3 cells with multivalent forms of an artificial ligand

Targeting and down-regulation of ErbB2, a member of EGF receptor family, is regarded as one of the key aspect for cancer treatment because it is often overexpressed in breast and ovarian cancer cells. Although natural ligands for ErbB2 have not been found, unlike other ErbB receptors, EC-1, a 20-amino acid circular peptide, has been shown to bind to ErbB2 as an artificial ligand. Previously we showed EC-1 peptide did not induce the internalization of ErbB2 in SK-BR-3 cells. In this report, we designed divalent and multivalent forms of EC-1 peptide with the Fc portion of the human IgG and bionanocapsule modified with ZZ-tag on its surface to improve the interaction with ErbB2. These forms showed higher affinity to ErbB2 than that of EC-1 monomer. Furthermore, prominent endosomal accumulation of ErbB2 occurred in SK-BR-3 cells when stimulated with EC-Fc ligand multivalently displayed on the surface of the bionanocapsule, whereas SK-BR-3 cells as themselves displayed stringent mechanism against ErbB2 internalization without stimulation. The multivalent form of EC-1 peptide appeared to internalize ErbB2 more efficiently than divalent form did. This internalization was unaffected by the inhibition of clathrin association, but inhibited when the cholesterol was depleted which explained either caveolar or GPI-AP-early endocytic compartment (GEEC) pathway. Because of the lack of caveolin-1 expression, caveolar machinery may be lost in SK-BR-3 cell line. Therefore, it is suggested that the multivalent form of EC-1 induces the internalization of ErbB2 through the GEEC pathway.     

Design, synthesis and pharmacological evaluation of cyclic mimetics of the insulin-like peptide 3 (INSL3) B-chain.

Insulin-like peptide 3 (INSL3) is a peptide hormone belonging to the relaxin-insulin superfamily of peptides that plays important roles in testes descent, oocyte maturation and the control of male germ cell apoptosis. These actions are mediated via a specific G-protein coupled receptor, LGR8. Previous structure-activity studies have shown that the key binding site of INSL3 is situated within its B-chain. Recent studies in our laboratory have led to the identification of a cyclic peptide mimetic 2 of the INSL3 B-chain, which we have shown to compete with the binding of [33P]-relaxin to LGR8 expressed in HEK293T cells, and to inhibit cAMP-mediated signaling in these cells, i.e. it is an antagonist of INSL3. In order to further define the structure-activity relationships of cyclic analogues of the INSL3 B-chain, we used a structure-based approach to design a series of cyclic, disulfide-constrained INSL3 B-chain mimetics. To do this, we first created a model of the 3D structure of INSL3 using the crystal structure of human relaxin as a template. This model of INSL3 was then used as a template to design a series of disulfide-constrained mimetics of the INSL3 B-chain. The peptides were synthesized by solid-phase peptide synthesis using pseudoproline dipeptides to improve the synthesis outcome. Of the seven prepared INSL3 B-chain mimetics, three compounds were found to have partial displacement activity, while four were able to completely displace [33P]-relaxin from LGR8, including compounds that were markedly shorter than compound 2. The best of these, mimetic 6, showed significantly greater affinity for LGR8 than compound 2, but still displayed around 1000-fold less affinity for LGR8 than native INSL3. Analysis of selected mimetics for their alpha-helical content using circular dichroism (CD) spectroscopy revealed that, generally, the mimetics showed less than expected helicity. The inability of the compounds to display true native INSL3 structure is likely contributing to their reduced receptor binding affinity. We are currently examining alternative INSL3 B-chain mimetics that might better present key receptor binding residues in the native INSL3-like conformation.     

Facile synthesis of peptide nucleic acids and peptide nucleic acid‐peptide conjugates on an automated peptide synthesizer

Peptide nucleic acids (PNAs) are DNA mimics with a neutral peptide backbone instead of the negatively charged sugar phosphates. PNAs exhibit several attractive features such as high chemical and thermal stability, resistance to enzymatic degradation, and stable binding to their RNA or DNA targets in a sequence‐specific manner. Therefore, they are widely used in molecular diagnosis of antisense‐targeted therapeutic drugs or probes and in pharmaceutical applications. However, the main hindrance to the effective use of PNAs is their poor uptake by cells as well as the difficult and laborious chemical synthesis. In order to achieve an efficient delivery of PNAs into cells, there are already many published reports of peptides being used for transport across the cell membrane. In this protocol, we describe the automated as well as cost‐effective semi‐automated synthesis of PNAs and PNA‐peptide constructs on an automated peptide synthesizer. The facile synthesis of PNAs will be helpful in generating PNA libraries usable, e.g. for high‐throughput screening in biomolecular studies. Efficient synthetic schemes, the automated procedure, the reduced consumption of costly reagents, and the high purity of the products are attractive features of the reported procedure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Piezoelectric property of bundled peptide nanotubes stapled by bis‐cyclic‐β‐peptide

Cyclic tetra‐β‐peptides (CP4s) and a bis‐CP4 were synthesized to prepare peptide nanotubes (PNTs) by molecular stacking of cyclic peptides. The addition of bis‐CP4 to the PNT preparation afforded PNT bundles increasing the direct and converse piezoelectiric coefficients, which is ascribable to bis‐CP4 stapling PNTs into the parallel alignment of PNT dipoles.     

Fmoc-based solid phase chemical synthesis of 71-meric neuregulin 1-beta1, an epidermal growth factor-like domain.

The human neuregulin 1-beta1 (NRG1-beta1, amino acid residues 176-246) was chemically synthesized by Fmoc-based solid phase peptide synthesis (SPPS) followed by folding in a redox buffer. The biological activity of the synthesized NRG1-beta1 was confirmed by ligand-induced tyrosine phosphorylation on Chinese hamster ovary (CHO) cells expressing ErbB-4.     

ErbB2 and EGFR are downmodulated during the differentiation of 3T3-L1 preadipocytes

The expression of receptors belonging to the epidermal growth factor receptor subfamily has been largely studied these last years in epithelial cells mainly as involved in cell proliferation and malignant progression. Although much work has focused on the role of these growth factor receptors in the differentiation of a variety of tissues, there is little information in regards to normal stromal cells. We investigated erbB2 expression in the murine fibroblast cell line Swiss 3T3L1, which naturally or hormonally induced undergoes adipocyte differentiation. We found that the Swiss 3T3-L1 fibroblasts express erbB2, in addition to EGFR, and in a quantity comparable to or even greater than the breast cancer cell line T47D. Proliferating cells increased erbB2 and EGFR levels when reaching confluence up to 4- and 10-fold, respectively. This expression showed a significant decrease when growth-arrested cells were stimulated to differentiate with dexamethasone and isobutyl-methylxanthine. Differentiated cells presented a decreased expression of both erbB2 and EGFR regardless of whether the cells were hormonally or spontaneously differentiated. EGF stimulation of serum-starved cells increased erbB2 tyrosine phosphorylation and retarded erbB2 migration in SDS-PAGE, suggesting receptor association and activation. Heregulin-alpha1 and -beta1, two EGF related factors, had no effect on erbB2 or EGFR phosphorylation. Although 3T3-L1 cells expressed heregulin, its specific receptors, erbB3 and erbB4, were not found. This is the first time in which erbB2 is reported to be expressed in an adipocytic cell line which does not depend on non EGF family growth factors (thyroid hormone, growth hormone, etc.) to accomplish adipose differentiation. Since erbB2 and EGFR expression were downmodulated as differentiation progressed it is conceivable that a mechanism of switching from a mitogenic to a differentiating signaling pathway may be involved, through regulation of the expression of these growth factor receptors.     

Down-modulation of erbB2 activity is necessary but not enough in the differentiation of 3T3-L1 preadipocytes

The high incidence of obesity-related pathologies, led to the study of the mechanisms involved in preadipose cell proliferation and differentiation. Here, we demonstrate that modulation of erbB2, plays a fundamental role during proliferation and adipogenic induction of preadipocytes. Using 3T3-L1 cells as model, we demonstrate that EGF (10 nM, 5 min) in addition to stimulate receptor tyrosine phosphorylation of both erbB2 and EGFR, is able to induce the heterodimer erbB2-EGFR. We treated proliferating 3T3-L1 cells with two inhibitors, AG 825 (IC(50) 0.35 microM, 54 times more selective for erbB2 than for EGFR, IC(50) 19 microM), and AG 879 (IC(50) of 1 microM for erbB2 versus 500 microM for EGFR). We found that both inhibited the proliferation on a dose-dependent basis, reaching a 30% maximal inhibition at 100 microM (P < 0.001) for AG825, and a 20% maximal inhibition at 10 microM (P < 0.001) for AG 879. These results involve erbB2 in 3T3-L1 proliferation. When studying the differentiation process, we found that the action of MIX-Dexa immediately activates MEK, JNK and p38 kinases. We observed that PD98059 and SP600125 (MEK-ERK and JNK inhibitors, respectively) added 1 h prior to the MIX-Dexa induction produced a decrease in erbB2 expression after 6 h, which is even greater than the one produced by the inducers, MIX-Dexa. This work supports erbB2 as a key factor in 3T3-L1 adipogenesis, acting mostly and not only during the proliferative phase but also during the differentiation through modulation of both its expression and activity.     

Micelle bound structure and DNA interaction of brevinin‐2‐related peptide,an antimicrobial peptide derived from frog skin

Brevinin‐2‐related peptide (BR‐II), a novel antimicrobial peptide isolated from the skin of frog, Rana septentrionalis, shows a broad spectrum of antimicrobial activity with low haemolytic activity. It has also been shown to have antiviral activity, specifically to protect cells from infection by HIV‐1. To understand the active conformation of the BR‐II peptide in membranes, we have investigated the interaction of BR‐II with the prokaryotic and eukaryotic membrane‐mimetic micelles such as sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC), respectively. The interactions were studied using fluorescence and circular dichroism (CD) spectroscopy. Fluorescence experiments revealed that the N‐terminus tryptophan residue of BR‐II interacts with the hydrophobic core of the membrane mimicking micelles. The CD results suggest that interactions with membrane‐mimetic micelles induce an α‐helix conformation in BR‐II. We have also determined the solution structures of BR‐II in DPC and SDS micelles using NMR spectroscopy. The structural comparison of BR‐II in the presence of SDS and DPC micelles showed significant conformational changes in the residues connecting the N‐terminus and C‐terminus helices. The ability of BR‐II to bind DNA was elucidated by agarose gel retardation and fluorescence experiments. The structural differences of BR‐II in zwitterionic versus anionic membrane mimics and the DNA binding ability of BR‐II collectively contribute to the general understanding of the pharmacological specificity of this peptide towards prokaryotic and eukaryotic membranes and provide insights into its overall antimicrobial mechanism. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Mad2 and BubR1 modulates tumourigenesis and paclitaxel response in MKN45 gastric cancer cells

Aneuploidy and chromosomal instability (CIN) are common features of gastric cancer (GC), but their contribution to carcinogenesis and antitumour therapy response is still poorly understood. Failures in the mitotic checkpoint induced by changes in expression levels of the spindle assembly checkpoint (SAC) proteins cause the missegregation of chromosomes in mitosis as well as aneuploidy. To evaluate the possible contribution of SAC to GC, we analyzed the expression levels of proteins of the mitotic checkpoint complex in a cohort of GC cell lines. We found that the central SAC proteins, Mad2 and BubR1, were the more prominently expressed members in disseminated GC cell lines. Silencing of Mad2 and BubR1 in MKN45 and ST2957 cells decreased their cell proliferation, migration and invasion abilities, indicating that Mad2 and BubR1 could contribute to cellular transformation and tumor progression in GC. We next evaluated whether silencing of SAC proteins could affect the response to microtubule poisons. We discovered that paclitaxel treatment increased cell survival in MKN45 cells interfered for Mad2 or BubR1 expression. However, apoptosis (assessed by caspase-3 activation, PARP proteolysis and levels of antiapoptotic Bcl 2-family members), the DNA damage response (assessed by H2Ax phosphorylation) and exit from mitosis (assessed by Cyclin B degradation and Cdk1 regulation) were activated equally between cells, independently of Mad2 or BubR1-protein levels. In contrast, we observed that the silencing of Mad2 or BubR1 in MKN45 cells showed the induction of a senescence-like phenotype accompanied by cell enlargement, increased senescence-associated β-galactosidase activity and increased IL-6 and IL-8 expression. In addition, the senescent phenotype is highly increased after treatment with PTX, indicating that senescence could prevent tumorigenesis in GC. In conclusion, the results presented here suggest that Mad2 and BubR1 could be used as prognostic markers of tumor progression and new pharmacological targets in the treatment for GC.     

Intranasal immunization with a peptide conjugated to Salmonella flagellin induces both systemic and mucosal peptide‐specific antibody responses in mice

In this study, the mucosal adjuvant activity of Salmonella flagellin as a carrier in a conjugate of EXP153–rFliC was investigated. EXP153–rFliC was made by conjugation of a synthetic B‐cell epitope peptide derived from Plasmodium falciparum exported protein‐1(EXP153) to recombinant phase 1 flagellin of Salmonella enterica serovar Typhimurium expressed in Escherichia coli (rFliC), and used to immunize BALB/c mice via intranasal instillation. It was found that robust EXP153‐specific serum IgG antibodies were induced without additional adjuvant. EXP153‐specific sIgA antibodies were also induced, these being detected in bronchoalveolar, nasal, vaginal and intestinal washes. These observations demonstrate that Salmonella flagellin as a carrier is an effective mucosal adjuvant in that its conjugated peptide induces antibody responses.     

N-Formylated humanin activates both formyl peptide receptor-like 1 and 2

We have discovered that humanin (HN) acts as a ligand for formyl peptide receptor-like 1 (FPRL1) and 2 (FPRL2). This discovery was based on our finding that HN suppressed forskolin-induced cAMP production in Chinese hamster ovary (CHO) cells expressing human FPRL1 (CHO-hFPRL1) or human FPRL2 (CHO-hFPRL2). In addition, we found that N-formylated HN (fHN) performed more potently as a ligand for FPRL1 than HN: in CHO-hFPRL1 cells, the effective concentration for the half-maximal response (EC(50)) value of HN was 3.5nM, while that of fHN was 0.012nM. We demonstrated by binding experiments using [(125)I]-W peptide that HN and fHN directly interacted with hFPRL1 on the membrane. In addition, we found that HN and fHN showed strong chemotactic activity for CHO-hFPRL1 and CHO-hFPRL2 cells. HN is known to have a protective effect against neuronal cell death. Our findings contribute to the understanding of the mechanism behind HN's function.     

Inhibition of HIV-1 infection by an intramolecular antisense peptide to T20 in gp160

Antisense amino acids are amino acids which can be translated from the corresponding anti-codons of a sense amino acid. Antisense peptides encoded by the noncoding DNA strand have a tendency to interact with each other. We have demonstrated that antisense peptide sequences are present intramolecularly, and these may contribute to the folding and maintenance of the tertiary structure of a protein. T20 is a synthetic peptide with an amino acid sequence in the gp41 of HIV-1 and has been demonstrated to be a potent inhibitor of HIV-1 infection. We searched for intramolecular peptide sequences which are antisense to portions of T20. A synthetic peptide (TA-1L) consisting of amino acids 84 to 97 of gp160, which contains an antisense peptide sequence (TA-1) to T20, was shown to inhibit HIV-1(IIIB) infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. The TA-1L site, which exists in the C1 domain of gp160, is highly homologous among strains of HIV-1, especially at TA-1 and in the amino acids flanking the C terminus. Although the TA-1 sites of 18 out of 30 HIV-1 strains were antisense to the T20 region, those of the remaining 12 strains, including HIV-1(MN), were not. However, TA-1L inhibited infection by HIV-1(MN), which has no antisense peptide in T20 corresponding to TA-1, although the inhibitory effect was weaker. TA-1L may thus also interfere with the gp160 interaction with CD4, which has an antisense sequence to TA-1.     

Nanofiber formation of amphiphilic cyclic tri‐β‐peptide

A novel amphiphilic cyclic peptide composed of two β‐glucosamino acids and one trans‐2‐aminocyclohexylcarboxylic acid was synthesized and investigated on assembly formation. The cyclic tri‐β‐peptide was self‐assembled into rodlike crystals or nanofibers depending on preparative conditions. The rodlike crystals showed a layer spacing of 4.8 Å along the long axis, and columnar spacings of 10.8 and 21.5 Å by electron diffraction analysis along the short axis. The former confirms the columnar structure upon molecular stacking, and the latter indicates triple bundle formation of the columnar assemblies. Fourier transform infrared (FT‐IR) measurement of the fibrous assembly showed formation of homogeneous hydrogen bonds among amide groups, also supporting the molecular stacking of cyclic β‐peptides. Straight nanofibers with uniform diameter were also uniquely obtained. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Design and mechanism of action of a novel bacteria-selective antimicrobial peptide from the cell-penetrating peptide Pep-1

Here, we report the successful design of a novel bacteria-selective antimicrobial peptide, Pep-1-K (KKTWWKTWWTKWSQPKKKRKV). Pep-1-K was designed by replacing Glu-2, Glu-6, and Glu-11 in the cell-penetrating peptide Pep-1 with Lys. Pep-1-K showed strong antibacterial activity against reference strains (MIC = 1-2 microM) of Gram-positive and Gram-negative bacteria as well as against clinical isolates (MIC = 1-8 microM) of methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. In contrast, Pep-1-K did not cause hemolysis of human erythrocytes even at 200 microM. These results indicate that Pep-1-K may be a good candidate for antimicrobial drug development, especially as a topical agent against antibiotic-resistant microorganisms. Tryptophan fluorescence studies indicated that the lack of hemolytic activity of Pep-1-K correlated with its weak ability to penetrate zwitterionic phosphatidylcholine/cholesterol (10:1, w/w) vesicles, which mimic eukaryotic membranes. Furthermore, Pep-1-K caused little or no dye leakage from negatively charged phosphatidylethanolamine/phosphatidylglycerol (7:3, w/w) vesicles, which mimic bacterial membranes but had a potent ability to cause depolarization of the cytoplasmic membrane potential of intact S. aureus cells. These results suggested that Pep-1-K kills microorganisms by not the membrane-disrupting mode but the formation of small channels that permit transit of ions or protons but not molecules as large as calcein.     

Efficient methodology for the cyclization of linear peptide libraries via intramolecular S-alkylation using Multipin solid phase peptide synthesis.

Methodology is described here for the efficient parallel synthesis and cyclization of linear peptide libraries using intramolecular S-alkylation chemistry in combination with Multipin solid phase peptide synthesis (Multipin SPPS). The effective use of this methodology was demonstrated with the synthesis of a 72-member combinatorial library of cyclic thioether peptide derivatives of the conserved four-residue structural motif DD/EXK found in the active sites of the five crystallographically defined orthodox type II restriction endonucleases, EcoRV, EcoRI, PvuII, BamHI and BglI.     

Synthetic peptide octarphin (TPLVTLFK), a selective agonist of nonopioid β‐endorphin receptor,stimulates nitric oxide synthesis in macrophages

Synthetic peptide octarphin (TPLVTLFK, a selective agonist of nonopioid β‐endorphin receptor) was able to activate in a dose‐dependent manner murine macrophages to express nitric oxide (NO) synthase and to produce NO. Octarphin required lipopolysacharide for the optimal induction of NO production. Octarphin‐dependent NO production was sensitive to inhibition by dexamethasone and the NO synthase specific inhibitor NG‐monomethyl‐l ‐arginine. In the concentration range of 1–1000 nM, octarphin increased the cyclic 3′,5′‐guanosine monophosphate (cGMP) content in macrophages stimulated with lipopolysacharide. The effect was dependent on the peptide concentration and was maximal at a concentration of 100 nM. Thus, octarphin stimulates both NO and cGMP production in macrophages. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Conformation of an Shc-derived phosphotyrosine-containing peptide complexed with the Grb2 SH2 domain

We have determined the structure of an Shc-derivedphosphotyrosine-containing peptide complexed with Grb2 SH2 based on intra-and intermolecular NOE correlations observed by a series of isotope-filteredNMR experiments using a PFG z-filter. In contrast to an extendedconformation of phosphotyrosine-containing peptides bound to Src, Syp andPLC SH2s, the Shc-derived peptide formed a turn at the +1 and +2positions next to the phosphotyrosine residue. Trp¹²¹, locatedat the EF1 site of Grb2 SH2, blocked the peptide binding in an extendedconformation. The present study confirms that eachphosphotyrosine-containing peptide binds to the cognate SH2 with a specificconformation, which gives the structural basis for the binding specificitybetween SH2s and target proteins.     

Computational analysis of molecular basis of 1:1 interactions of NRG-1beta wild-type and variants with ErbB3 and ErbB4

The neuregulin/ErbB system is a growth factor/receptor cascade that has been proven to be essential in the development of the heart and the sympathetic nervous system. However, the basis of the specificity of ligand-receptor recognition remains to be elucidated. In this study, the structures of NRG-1beta/ErbB3 and NRG-1beta/ErbB4 complexes were modeled based on the available structures of the homologous proteins. The binding free energies of NRG-1beta to ErbB3 and ErbB4 were calculated using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) computational method. In addition, computational alanine-scanning mutagenesis was performed in the binding site of NRG-1beta and the difference in the binding free energies between NRG-1beta mutants and the receptors was calculated. The results specify the contribution of each residue at the interaction interfaces to the binding affinity of NRG-1beta with ErbB3 and ErbB4, identifying several important interaction residue pairs that are in agreement with previously acquired experimental data. This indicates that the presented structural models of NRG-1beta/ErbB3 and NRG-1beta/ErbB4 complexes are reliable and could be used to guide future studies, such as performing desirable mutations on NRG-1beta to increase the binding affinity and selectivity to the receptor and discovering new therapeutic agents for the treatment of heart failure.     

A Dde resin based strategy for inverse solid-phase synthesis of amino terminated peptides, peptide mimetics and protected peptide intermediates.

This report describes a Dde resin based attachment strategy for inverse solid-phase peptide synthesis (ISPPS). This attachment strategy can be used for the synthesis of amino terminated peptides with side chains and the carboxyl terminus either protected or deprotected. Amino acid t-butyl esters were attached through their free amino group to the Dde resin. The t-butyl carboxyl protecting group was removed by 50% TFA, and inverse peptide synthesis cycles performed using an HATU/TMP based coupling method. Protected peptides were cleaved from the resin with dilute hydrazine. Side chain protecting groups could then be removed by treatment with TFMSA/TFA. The potential of this approach was demonstrated by the synthesis of several short protected and unprotected peptides in good yield and with low epimerization. Its potential for peptide mimetic synthesis was demonstrated by the synthesis of two peptide trifluoromethylketones.