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1.
Hemolysin E (HlyE), a pore-forming protein-toxin and a potential virulence factor of Escherichia coli, exhibits cytotoxic activity to mammalian cells. However, very little is known about how the different individual segments contribute in the toxic activity of the protein. Toward this end, the role of a 33-residue segment comprising the amino acid region 88 to 120, which contains the putative transmembrane domain in the tail region of HlyE has been addressed in the toxic activity of the protein-toxin by characterizing the related wild type and mutant peptides and the whole protein. Along with the 33-residue wild type peptide, H-88, two mutants of the same size were synthesized; in one mutant a conserved valine at 89th position was replaced by aspartic acid and in the other both glycine and valine at the 88th and 89th positions were substituted by aspartic acid residues. These mutations were also incorporated in the whole toxin HlyE. Results showed that only H-88 but not its mutants permeabilized both lipid vesicles and human red blood cells (hRBCs). Interestingly, while H-88 exhibited a moderate lytic activity to human red blood cells, the mutants were not active. Drastic reduction in the depolarization of hRBCs and hemolytic activity of the whole toxin HlyE was also observed as a result of the same double and single amino acid substitution in it. The results indicate an important role of the amino acid segment 88-120, containing the putative transmembrane domain of the tail region of the toxin in the toxic activity of hemolysin E.  相似文献   

2.
Hemolysin E (HlyE) is a 34 kDa protein toxin, recently isolated from a pathogenic strain of Escherichia coli, which is believed to exert its toxic activity via formation of pores in the target cell membrane. With the goal of understanding the involvement of different segments of hemolysin E in the membrane interaction and assembly of the toxin, a conserved, amphipathic leucine zipper-like motif has been identified. In order to evaluate the possible structural and functional roles of this segment in HlyE, a 30-residue peptide (H-205) corresponding to the leucine zipper motif (amino acid 205-234) and two mutant peptides of the same size were synthesized and labeled by fluorescent probes at their N termini. The results show that the wild-type H-205 binds to both zwitterionic (PC/Chol) and negatively charged (PC/PG/Chol) phospholipid vesicles and also self-assemble therein. Detailed membrane-binding experiments revealed that this synthetic motif (H-205) formed large aggregates and inserted into the bilayer of only negatively charged lipid vesicles but not of zwitterionic membrane. Although both the mutants bound to zwitterionic and negatively charged lipid vesicles, neither of them inserted into the lipid bilayers nor assembled in any of these lipid vesicles. Furthermore, H-205 adopted a significant helical structure in membrane mimetic environments and induced the permeation of monovalent ions and release of entrapped calcein across the phospholipid vesicles more efficiently than the mutant peptides. The results presented here indicate that this H-205 (amino acid 205-234) segment may be an important structural element in hemolysin E, which could play a significant role in the binding and assembly of the toxin in the target cell membrane and its destabilization.  相似文献   

3.
To find out the sequence requirement of the H-205 peptide, containing an amphipathic leucine zipper motif corresponding to the amino acid (a.a.) region 205-234 of hemolysin E (HlyE) to induce efficient permeation in zwitterionic lipid vesicles, the peptide was extended at the N-terminal after the addition of seven amino acids from the predicted transmembrane region in the head domain of the protein-toxin. The new peptide, H-198 (a.a. 198-234) and a scrambled mutant peptide of the same size were synthesized, fluorescently labeled and characterized functionally and structurally. The results showed that H-198 induced significantly higher permeation in the zwitterionic PC/Chol lipid vesicles than its shorter version, H-205. H-198 formed large aggregates in the PC/Chol vesicles unlike H-205 and also adopted more helical structure in the membrane mimetic environments compared to that of H-205. Fluorescence energy transfer experiments by flow cytometry indicated that only H-198 but not its mutant or H-205 oligomerized in the zwitterionic lipid vesicles, while in the negatively charged lipid vesicles both H-198 and H-205 formed oligomeric assembly. The results suggest a probable role of the hydrophobic residues of the head domain of HlyE in inducing permeability in the zwitterionic lipid vesicles by the peptide derived from the a.a. 198-234 of the toxin.  相似文献   

4.
To find out the sequence requirement of the H-205 peptide, containing an amphipathic leucine zipper motif corresponding to the amino acid (a.a.) region 205-234 of hemolysin E (HlyE) to induce efficient permeation in zwitterionic lipid vesicles, the peptide was extended at the N-terminal after the addition of seven amino acids from the predicted transmembrane region in the head domain of the protein-toxin. The new peptide, H-198 (a.a. 198-234) and a scrambled mutant peptide of the same size were synthesized, fluorescently labeled and characterized functionally and structurally. The results showed that H-198 induced significantly higher permeation in the zwitterionic PC/Chol lipid vesicles than its shorter version, H-205. H-198 formed large aggregates in the PC/Chol vesicles unlike H-205 and also adopted more helical structure in the membrane mimetic environments compared to that of H-205. Fluorescence energy transfer experiments by flow cytometry indicated that only H-198 but not its mutant or H-205 oligomerized in the zwitterionic lipid vesicles, while in the negatively charged lipid vesicles both H-198 and H-205 formed oligomeric assembly. The results suggest a probable role of the hydrophobic residues of the head domain of HlyE in inducing permeability in the zwitterionic lipid vesicles by the peptide derived from the a.a. 198-234 of the toxin.  相似文献   

5.
We have recently demonstrated that a 37-amino acid peptide corresponding to the cytoplasmic domain of the natriuretic peptide receptor C (NPR-C) inhibited adenylyl cyclase activity via pertussis toxin (PT)-sensitive G(i) protein. In the present studies, we have used seven different peptide fragments of the cytoplasmic domain of the NPR-C receptor with complete, partial, or no G(i) activator sequence to examine their effects on adenylyl cyclase activity. The peptides used were KKYRITIERRNH (peptide 1), RRNHQEESNIGK (peptide 2), HRELREDSIRSH (peptide 3), RRNHQEESNIGKHRELR (peptide 4), QEESNIGK (peptide X), ITIERRNH (peptide Y), and ITIYKKRRNHRE (peptide Z). Peptides 1, 3, and 4 have complete G(i) activator sequences, whereas peptides 2 and Y have partial G(i) activator sequences with truncated carboxyl or amino terminus, respectively. Peptide X has no structural specificity, whereas peptide Z is the scrambled peptide control for peptide 1. Peptides 1, 3, and 4 inhibited adenylyl cyclase activity in a concentration-dependent manner with apparent K(i) between 0.1 and 1 nm; however, peptide 2 inhibited adenylyl cyclase activity with a higher K(i) of about 10 nm, and peptides X, Y, and Z were unable to inhibit adenylyl cyclase activity. The maximal inhibitions observed were between 30 and 40%. The inhibition of adenylyl cyclase activity by peptides 1-4 was absolutely dependent on the presence of guanine nucleotides and was completely attenuated by PT treatment. In addition, the stimulatory effects of isoproterenol, glucagon, and forskolin on adenylyl cyclase activity were inhibited to different degrees by these peptides. These results suggest that the small peptide fragments of the cytoplasmic domain of the NPR-C receptor containing 12 or 17 amino acids were sufficient to inhibit adenylyl cyclase activity through a PT-sensitive G(i) protein. The peptides having complete structural specificity of G(i) activator sequences at both amino and carboxyl termini were more potent to inhibit adenylyl cyclase activity as compared with the peptides having a truncated carboxyl terminus, whereas the truncation of the amino-terminal motif completely attenuates adenylyl cyclase inhibition.  相似文献   

6.
Recent evidence has demonstrated a protein kinase C (PKC)-dependent step in cytotoxic T lymphocyte activation. Here, we examined the influence of PKC in the lytic response of human NK cells to K562, an NK-sensitive tumor target cell. We used the known protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and HA1004. H-7 caused a dose-related inhibition of NK cell-mediated cytolysis (CMC) when the inhibitor was present throughout the course of the 3-h chromium release assay. The 50% inhibitory concentration for H-7 was 7 microM. In contrast, HA1004, which exerts a greater inhibitory effect on cyclic nucleotide-dependent protein kinases than PKC, had no effect on NK-CMC. The suppression of NK-CMC by H-7 was not due to inhibition of binding of the effector cells to target cells and could be reversed by the addition of PMA. H-7 was most effective in abrogating NK-CMC when added to the assay within the first 30 min and treatment of the effector and target cells with H-7 resulted in no loss of NK-CMC. Because nearly 50% of the normal NK lytic activity had taken place by 30 min, this suggested that H-7 inhibited an early event. H-7 exerted a dose-related suppression of antibody-dependent cell-mediated cytotoxicity (ADCC) suggesting that NK-CMC and ADCC share the utilization of PKC, however, HA1004 did not inhibit ADCC. Treating NK cells with IL-2 or IFN-beta did not overcome the inhibition of NK-CMC by H-7. In this study, we have thus demonstrated the presence of a PKC-dependent step in NK-CMC and ADCC.  相似文献   

7.
8.
9.
Neurotensin, bradykinin and somatostatin inhibited in a time- and concentration-dependent manner prostaglandin E1- or forskolin-stimulated cAMP production in neuroblastoma N1E115 cells. Cell treatment with 1 microgram/ml pertussis toxin for 6 hours reversed the inhibition elicited by peptides after short incubation periods (less than or equal to 1 min) but, in contrast, had no effect after longer incubation periods (greater than or equal to 3 min). Fluoroaluminate also inhibited prostaglandin E1-stimulated cAMP production in N1E115 cells, and this effect was not reversed by pertussis toxin. The 6 hour treatment with pertussis toxin was shown to be sufficient to ADP ribosylate virtually all of the 41 kD protein substrate corresponding to the alpha subunit of Gi. Protein kinase C activation with phorbol ester did not inhibit basal or stimulated cAMP production. Our data point to the existence of both pertussis toxin sensitive and insensitive mechanisms of neuropeptide-mediated inhibition of cAMP formation in N1E115 cells. The toxin insensitive response is not mediated by protein kinase C. The possibility is discussed that it results from the activation of a pertussis toxin insensitive G protein.  相似文献   

10.
11.
Although BMAP-28 is a potent cathelicidin-derived bovine antimicrobial peptide, its cytotoxic activity against the human and other mammalian cells is of concern for converting it into a novel antimicrobial drug. We have identified a short leucine and isoleucine zipper sequences at the N- and C-terminals of BMAP-28, respectively. To understand the possible role of these structural elements in BMAP-28, a number of alanine-substituted analogs were designed, synthesized and characterized along with the wild-type peptide. The substitution of amino acids at single or multiple ‘a’ position(s) of these structural motifs by alanine showed significant effects on the cytotoxic activity of the molecule on the human red blood cells (hRBCs) and 3T3 cells without showing much effects on their MIC values against the selected bacteria. BMAP-28 and all its analogs depolarized the Escherichia coli cells with almost equal efficacy. In contrast, the alanine-substituted analogs of BMAP-28 depolarized hRBCs much less efficiently than the parent molecule. Results further showed that BMAP-28 assembled appreciably onto the live E. coli and hRBC. However, the selected less toxic analogs of BMAP-28 although assembled as good as the parent molecule onto the live E. coli cells, their assembly onto the live mammalian hRBCs was much weaker as compared to that of the wild-type molecule. Looking at the remarkable similarity with the data presented in our previous work on melittin, it appears that probably the heptad repeat sequence possesses a general role in maintaining the cytotoxicity of the antimicrobial peptides against the mammalian cells and assembly therein.  相似文献   

12.
The physical association of HLA class I or H-2 molecules with 36 HIV-1 Nef synthetic peptides was studied using a direct peptide binding assay (PBA) in solid phase. To assess the functional significance of the PBA results, the Nef peptides were also tested for their ability to inhibit the lytic activity of human or murine CTL. The PBA results showed that seven partly overlapping regions of the Nef protein contained MHC binding peptides (4-18, 46-67, 73-94, 100-128, 126-155, 182-198, and 192-206). Five of these seven regions included all the already described epitopes recognized by CD8+ human CTL. The two other regions, 4-18 and 46-67, are not yet described as antigenic for human CD8+ cells but they are located in the N-terminal part of Nef that was previously described as being stimulator for rat or chimpanzee CD4+ cells. Altogether, it can be concluded that 1) In virtually 100% of the cases, the PBA is capable to detect known antigenic peptides recognized by CTL. 2) The PBA and the functional inhibition assay provide similar results, supporting the functional significance of PBA results. 3) The PBA is easy to handle on a large scale, using multiple peptide and several MHC molecules, so that it can be used as a routine method for prevision of possibly epitopic sequences. 4) Systematic studies of peptides issued from the whole sequence of a given protein allow to map polyepitopic areas that are probably the most interesting parts of proteins for a vaccine purpose.  相似文献   

13.
Exposure to the tiglian 12-O-tetradecanoylphorbol-13-acetate (TPA) represents one of the most efficient and widely used protocols for inducing Epstein-Barr virus (EBV)-infected cells from latent into lytic cycle. Since TPA is both a potent tumor promoter and a potent activator of the cellular protein kinase C (PKC), we sought to determine whether either of these activities was closely linked to EBV lytic cycle induction. A panel of TPA structural analogs, encompassing tiglians with different spectra of biological activities, was assayed on a number of EBV-positive B-lymphoid cell lines. Lytic cycle induction correlated with the capacity to activate PKC, not with tumor promoter status; some nonpromoting tiglians were as efficient as TPA in inducing lytic cycle antigen expression. We then sought more direct evidence for an involvement of PKC in the induction process. In initial experiments, 1-(5-isoquinolinyl sulphonyl)-2-methylpiperazine (H-7), the best available pharmacological inhibitor of PKC, completely blocked the induction of the lytic cycle by TPA and its active analogs. This is consistent with, but does not prove, a requirement for active PKC in the induction process, since H-7 targets PKC preferentially but also has some effects on other kinases. We therefore turned to the synthetic pseudosubstrate peptide PKC(19-36) as a means of specific PKC inhibition and to the closely related but inactive peptide PKC(19-Ser-25-36) as a control. Using the technique of scrape loading to deliver the peptides into cells of an adherent EBV-positive target line, we found that the pseudosubstrate peptide PKC(19-36) completely and specifically blocked tiglian-induced entry of the cells into the lytic cycle. The evidence both from TPA analogs and from enzyme inhibition studies therefore indicates that the pathway linking TPA treatment to lytic cycle induction involves active PKC. Interestingly, inhibition of PKC had no effect upon the spontaneous entry into lytic cycle which occurs in naturally productive cell lines, suggesting that spontaneous entry is signalled by another route.  相似文献   

14.
Nonenveloped viruses often invade membranes by exposing hydrophobic or amphipathic peptides generated by a proteolytic maturation step that leaves a lytic peptide noncovalently associated with the viral capsid. Since multiple copies of the same protein form many nonenveloped virus capsids, it is unclear if lytic peptides derived from subunits occupying different positions in a quasi-equivalent icosahedral capsid play different roles in host infection. We addressed this question with Nudaurelia capensis omega virus (NωV), an insect RNA virus with an icosahedral capsid formed by protein α, which undergoes autocleavage during maturation, producing the lytic γ peptide. NωV is a unique model because autocatalysis can be precisely initiated in vitro and is sufficiently slow to correlate lytic activity with γ peptide production. Using liposome-based assays, we observed that autocatalysis is essential for the potent membrane disruption caused by NωV. We observed that lytic activity is acquired rapidly during the maturation program, reaching 100% activity with less than 50% of the subunits cleaved. Previous time-resolved structural studies of partially mature NωV particles showed that, during this time frame, γ peptides derived from the pentamer subunits are produced and are organized in a vertical helical bundle that is projected toward the particle surface, while identical polypeptides in quasi-equivalent subunits are produced later or are in positions inappropriate for release. Our functional data provide experimental support for the hypothesis that pentamers containing a central helical bundle, observed in different nonenveloped virus families, are a specialized lytic motif.  相似文献   

15.
The isoquinolinesulfonamide PKC inhibitors H-7 and H-8 inhibit primary, in vivo generated cytotoxic T lymphocyte (CTL) activity by 50% at concentrations approximating their reported Ki values for PKC, 6 uM and 15 uM respectively. However, a greater than ten-fold higher concentration of H-7 (100 uM) is required to reduce secondary or clone 8F CTL-mediated cytotoxicity by 50%. At this concentration H-7 is also reported to inhibit calmodulin (CaM)-dependent enzymes. To distinguish between the effect of 100 uM H-7 on PKC versus CaM the napthalenesulfonamide CaM antagonist W-7 was investigated. W-7 inhibited primary, secondary and clone 8F CTL-mediated cytolysis by 50% near its reported Ki value for CaM-dependent kinase activity, 12 uM. We conclude that W-7 and 100 uM H-7 reduce cytolysis by inhibiting CaM-dependent reactions and not PKC. Thus, these findings indicate that primary killers require both PKC- and CaM-dependent activation pathways for lethal hit delivery, whereas highly lytic cultured CTL use only one pathway dependent upon CaM.  相似文献   

16.
A specific stimulation of tubulin tyrosinolation in human neutrophils (PMNs) is induced by the synthetic peptide chemoattractant N-formylmethionylleucylphenylalanine (fMet-Leu-Phe), and this stimulation is closely associated with activation of the NADPH oxidase-mediated respiratory burst (Nath, J., and Gallin, J. I. (1983) J. Clin. Invest. 71, 1273-1281). In contrast, along with tubulin tyrosinolation, a distinctly different respiratory burst-associated random posttranslational incorporation of tyrosine into multiple PMN proteins is observed in PMNs stimulated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) or sn-1,2-dioctanoylglycerol (DAG). In studies exploring the mechanism(s) of signal transduction for these distinct neutrophil responses, we found that the fMet-Leu-Phe-induced stimulation of tubulin tyrosinolation in PMNs and in differentiated HL-60 cells is completely blocked by pertussis toxin, while the PMA-induced random incorporation of tyrosine is not inhibited. We also found that expression of the fMet-Leu-Phe-mediated stimulation of tubulin tyrosinolation in HL-60 cells is correlated with increases in the specific activity of protein kinase C and with the acquisition of respiratory burst activity which occur during induced myeloid maturation of these cells. Furthermore, both the fMet-Leu-Phe-induced stimulation of tubulin tyrosinolation and the PMA or DAG-induced random posttranslational incorporation of tyrosine into multiple proteins in activated neutrophils, were found to be reversibly inhibited (greater than 70%) by the protein kinase inhibitors 1-(5-isoquinolinesulfonyl)piperazine (C-I) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), in parallel with inhibition of superoxide (O2-) generation. In related studies, we also found that fMet-Leu-Phe-stimulated O2- production is comparably inhibited by C-I and H-7, but in a highly temperature-dependent manner. Inhibition was observed only when C-I or H-7 is added to PMNs at physiologic temperature, i.e. 37 degrees C. Interestingly, inhibition of the PMA-induced O2- generation by C-I or H-7 was not found to be similarly temperature-dependent. Considered together, these findings argue against the suggestion that there is a protein kinase C-independent pathway for activation of the respiratory burst in neutrophils stimulated with N-formyl peptides.  相似文献   

17.
Leukocyte function associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) have been shown to be critical for adhesion process and immune response. Modulation or inhibition of the interaction between LFA-1/ICAM-1 interactions can result in therapeutic effects. Our group and others have shown that peptides derived from ICAM-1 or LFA-1 inhibit adhesion in a homotypic T-cell adhesion assay. It is likely that the peptides derived from ICAM-1 bind to LFA-1 and peptides derived from LFA-1 bind to ICAM-1 and inhibit the adhesion interaction. However, there are no concrete experimental evidence to show that peptides bind to either LFA-1 or ICAM-1 and inhibit the adhesion. Using NMR, CD and docking studies we have shown that an LFA-1 derived peptide binds to soluble ICAM-1. Docking studies using "autodock" resulted in LFA-1 peptide interacting with the ICAM-1 protein near Glu34. The proposed model based on our experimental data indicated that the LFA-1 peptide interacts with the protein via three intermolecular hydrogen bonds. Hydrophobic interactions also play a role in stabilizing the complex.  相似文献   

18.
Animal peptide antibiotics are thought to mediate their cytotoxic and growth inhibitory action on bacteria, fungi, and cancer cells through a membrane-targeted mechanism. Although the membrane interactions of the peptide antibiotics and their penetration through the membranes have been studied in several models, the precise chain of events leading to cell death or growth arrest is not established yet. In this study we used in vitro kinase assays followed by imaging analyses to examine the effect of human cationic antimicrobial peptide ECAP on the activity of the protein kinases. We report that HPLC-grade ECAP is responsible for inhibition of EGFR autophosphorylation in plasma membrane fractions obtained from A-431 cells. The activity of ECAP is concentration dependent with a half-inhibitory concentration in the range of 0.1-0.2 microM. Marked decrease in autophosphorylation of immunoprecipitated non-receptor protein kinases belonging to different families, namely PKCmu, Lyn and Syk, is observed in the presence of as little as 0.2 microM of the peptide. Among the examined non-receptor protein kinases PKCmu was the most sensitive to the inhibitory action of ECAP, whereas Syk was inhibited least of all. ECAP exerted no detectable cytotoxicity on non-nucleate animal cells at concentrations up to 3 microM. The capability of ECAP to inhibit protein kinases at concentrations, that are at least 10 fold lower than antibacterial and cytotoxic ones, suggests that the protein kinases are possible intracellular targets for antimicrobial peptides. We suppose that inhibition of the protein kinases may provide a mechanism for the action of cationic antimicrobial peptides on host cells including tumour cells.  相似文献   

19.
The cytotoxic test system for Shigella shigae toxin was improved and used to study the stability of the toxin to various pH values, temperature, and chemicals. Inhibition of protein synthesis is the first demonstrable effect in cells treated with Shigella toxin. This inhibition appears to be at the level of peptide chain elongation. An inhibition effect on cell-free protein synthesis is exhibited by toxin pretreated first with trypsin and then with dithiothreitol and 8 M urea or 1% sodium dodecyl sulfate. Ribosomes treated with toxin or its A1 fragment had lost most of their ability to polymerize [14C]phenylalanine in a poly(U)-dependent cell-free system. Salt-washed ribosomes in simple buffered solutions were inactivated at a rate of at least 40 ribosomes/(min) (A1 fragment). Addition of antitoxin immediately stopped further inactivation, but it did not reactivate the inactivated ribosomes. 60 S ribosomal subunits from toxin-treated ribosomes had a marked reduction in ability to support polyphenylanine synthesis, whereas 40 S subunits from toxin-treated ribosomes retained their activity. Toxin-treated ribosomes retained their ability to incorporate [3H]puromycin into growing peptide chains, indicating that the peptide bond formation is not the function inhibited.  相似文献   

20.
The HIV-1 Integrase protein (IN) mediates the integration of the viral cDNA into the host genome. IN is an emerging target for anti-HIV drug design, and the first IN-inhibitor was recently approved by the FDA. We have developed a new approach for inhibiting IN by "shiftides": peptides derived from its cellular binding protein LEDGF/p75 that inhibit IN by shifting its oligomerization equilibrium from the active dimer to an inactive tetramer. In addition, we described two peptides derived from the HIV-1 Rev protein that interact with IN and inhibit its activity in vitro and in cells. In the current study, we show that the Rev-derived peptides also act as shiftides. Analytical gel filtration and cross-linking experiments showed that IN was dimeric when bound to the viral DNA, but tetrameric in the presence of the Rev-derived peptides. Fluorescence anisotropy studies revealed that the Rev-derived peptides inhibited the DNA binding of IN. The Rev-derived peptides inhibited IN catalytic activity in vitro in a concentration-dependent manner. Inhibition was much more significant when the peptides were added to free IN before it bound the viral DNA than when the peptides were added to a preformed IN-DNA complex. This confirms that the inhibition is due to the ability of the peptides to shift the oligomerization equilibrium of the free IN toward a tetramer that binds much weaker to the viral DNA. We conclude that protein-protein interactions of IN may serve as a general valuable source for shiftide design.  相似文献   

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