De novo designed cyclic cationic peptides as inhibitors of plant pathogenic bacteria

Head-to-tail cyclic peptides of 4-10 residues consisting of alternating hydrophilic (Lys) and hydrophobic (Leu and Phe) amino acids were synthesized and tested against the economically important plant pathogenic bacteria Erwinia amylovora, Xanthomonas vesicatoria and Pseudomonas syringae. The antibacterial activity, evaluated as the minimal inhibitory concentration (MIC), the cytotoxicity against human red blood cells and stability towards protease degradation were determined. The influence of cyclization, ring size, and replacement of l-Phe with d-Phe on antibacterial and hemolytic activities was studied and correlated with the degree of structuring and hydrophobicity. Our results showed that linear peptides were inactive against the three bacteria tested. Cyclic peptides were active only toward X. vesicatoria and P. syringae, being c(KLKLKFKLKQ) (BPC10L) the most active peptide with MIC values of 6.25 and 12.5 microM, respectively. The improved antibacterial activity of cyclic peptides compared to their linear counterparts was associated to an increase of the hydrophobicity, represented as RP-HPLC retention time (t(R)), and secondary structure content which are related to an enhanced amphipathicity. A decrease of antibacterial and hemolytic activities was observed when a d-Phe was introduced into the cyclic sequences, which was attributed to their low amphipathicity as shown by their low secondary structure content and low t(R). The small size, simple structure, bactericidal effect, and stability to protease degradation of the best peptides make them potential candidates for the development of effective antibacterial agents for use in plant protection.     

Control of the reactivation kinetics of homodimeric triosephosphate isomerase from unfolded monomers

Homodimeric triosephosphate isomerases from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have markedly similar catalytic properties and 3-D structures; their overall amino acid sequence identity is 68% and 85% in their interface residues. Nonetheless, active dimer formation from guanidinium chloride unfolded monomers is faster and more efficient in TcTIM than in TbTIM. The enzymes thus provide a unique opportunity for exploring the factors that control the formation of active dimers. The kinetics of reactivation at different protein concentrations showed that the process involved three reactions: monomer folding, association of folded monomers, and a transition from inactive to active dimers. The rate constants of the reactions indicated that, at relatively low protein concentrations, the rate-limiting step of reactivation was the association reaction; at high protein concentrations the transition of inactive to active dimers was rate limiting. The rates of the latter two reactions were higher in TcTIM than in TbTIM. Studies with a mutant of TcTIM that had the interface residues of TbTIM showed that the association rate constant was similar to that of TbTIM. However, the rate of the transition from inactive to active dimers was close to that of TcTIM; thus, this transition depends on the noninterfacial portion of the enzymes. When unfolded monomers of TcTIM and TbTIM were allowed to reactivate together, TcTIM, the hybrid, and TbTIM were formed in a proportion of 1:0.9:0.2. This distribution suggests that, in the hybrid, the characteristics of the TcTIM monomers influence the properties of TbTIM monomers.     

Primary structure of the human elafin precursor preproelafin deduced from the nucleotide sequence of its gene and the presence of unique repetitive sequences in the prosegment.

The human elafin gene was cloned and its entire nucleotide sequence was determined to deduce the amino acid sequence for the precursor of elafin, an elastase-specific inhibitor. The gene spans approximately 1.7 kb and is divided into 3 exons. The gene product preproelafin consists of 117 amino acids: the initiator Met, a putative 25-amino acid signal peptide, a pro-sequence of about 34 amino acids, and the C-terminal 57 amino acids for mature elafin. Possible covalent clotting of the prosegment and its physiological significance have been pointed out based on a remarkable sequence similarity between the pro-sequence and the guinea pig seminal clotting protein SVP-1.     

Structural requirements for activity of the pheromones of Ustilago hordei

Ustilago hordei, the cause of barley-covered smut, initiates mating with pheromones. Gene sequence analysis suggested that these pheromones, Uhmfa1 and Uhmfa2, would be farnesylated peptides. Although isolation of mating-type-specific activity was rarely possible, chromatographic separations of culture supernatants yielded fractions that stimulated or inhibited mating. Based on predicted amino acid sequences and mass spectra of stimulating fractions, a series of pheromone analogs were synthesized and their activities were determined. Underivatized Uhmfa1 (PGKSGSGLGYSTC) or Uhmfa2 (EGKGEPAPYC) peptides were inactive, while peptides that were farnesylated and/or methyl esterified specifically induced conjugation tubes by cells of the opposite mating type. Uhmfa1 truncated from the amino terminus beyond the lysine lost activity, while truncated Uhmfa2 remained partially active. In mating bioassays, a pheromone concentration-dependent default mating response was observed. In competition studies, shorter Uhmfa1 peptides lacking pheromone activity inhibited activity of full-length peptides most effectively when both had the same functional groups.     

Protease inhibitors derived from elafin and SLPI and engineered to have enhanced specificity towards neutrophil serine proteases

The secretory leukocyte protease inhibitor (SLPI), elafin, and its biologically active precursor trappin‐2 are endogeneous low‐molecular weight inhibitors of the chelonianin family that control the enzymatic activity of neutrophil serine proteases (NSPs) like elastase, proteinase 3, and cathepsin G. These inhibitors may be of therapeutic value, since unregulated NSP activities are linked to inflammatory lung diseases. However SLPI inhibits elastase and cathepsin G but not proteinase 3, while elafin targets elastase and proteinase 3 but not cathepsin G. We have used two strategies to design polyvalent inhibitors of NSPs that target all three NSPs and may be used in the aerosol‐based treatment of inflammatory lung diseases. First, we fused the elafin domain with the second inhibitory domain of SLPI to produce recombinant chimeras that had the inhibitory properties of both parent molecules. Second, we generated the trappin‐2 variant, trappin‐2 A62L, in which the P1 residue Ala is replaced by Leu, as in the corresponding position in SLPI domain 2. The chimera inhibitors and trappin‐2 A62L are tight‐binding inhibitors of all three NSPs with subnanomolar K_is, similar to those of the parent molecules for their respective target proteases. We have also shown that these molecules inhibit the neutrophil membrane‐bound forms of all three NSPs. The trappin‐2 A62L and elafin‐SLPI chimeras, like wild‐type elafin and trappin‐2, can be covalently cross‐linked to fibronectin or elastin by a tissue transglutaminase, while retaining their polypotent inhibition of NSPs. Therefore, the inhibitors described herein have the appropriate properties to be further evaluated as therapeutic anti‐inflammatory agents.     

Border sequences of Medicago truncatula CLE36 are specifically cleaved by endoproteases common to the extracellular fluids of Medicago and soybean

CLE (CLAVATA3/ESR-related) peptides are developmental regulators that are secreted into the apoplast. Little is known about the role of the sequences that flank CLE peptides in terms of their biological activity or how they are targeted by proteases that are known to liberate the final active CLE peptides from their precursor sequences. The biological activity of Medicago truncatula CLE36, which possesses broadly conserved border sequences flanking the putative final active CLE36 peptide product, was assessed. Using in vitro root growth assays and an in vitro root and callus formation assay it is shown that CLE36 peptides of different lengths possess differential biological activities. Using mass spectrometry, Glycine max and Medicago extracellular fluids were each shown to possess an endoproteolytic activity that recognizes and cleaves at border sequences in a synthetic 31 amino acid CLE36 'propeptide bait' to liberate biologically active peptide products. Inhibitor studies suggest that a subtilisin, in combination with a carboxypeptidase, liberated and trimmed CLE36, respectively, to form biologically relevant 11-15 amino acid cleavage products. The 15 amino acid cleavage product is more biologically potent on Arabidopsis than shorter or longer CLE peptides. In situ hybridization shows that the soybean orthologue of CLE36 (GmCLE34) is expressed in the provascular tissue. The results suggest that secreted subtilisins can specifically recognize the border sequences of CLE36 propeptides and liberate biologically active cleavage products. These secreted proteases may affect the stability and biological activity of CLE peptides in the apoplast or be involved in CLE36 processing.     

Structure prediction for peptides capable of inducing antibody formation in mice

A simple method for the sequence prediction of peptides capable of the in vivo stimulation of antibody production in mice without conjugation with protein carriers was proposed on the basis of literature data on the structure of T-helper epitopes active in vivo. According to this approach, a potentially active peptide should contain a nine-membered sequence with a hydrophobic amino acid residue in the first position and a positively charged residue in the ninth position. The efficiency of this approach was confirmed by the presence of such sequences in the previously described synthetic peptides with immune activities, by the application of this approach to the choice of immunogenic fragments within the sequences of various proteins that exhibited further the specific activity, and by the construction of immunogenic peptides on the basis of inactive natural sequences.     

Alpha-fetoprotein-derived antiestrotrophic octapeptide

Alpha-fetoprotein (AFP) is a major serum protein produced during fetal development. Experimental findings suggest that AFP has antiestrotrophic activity and that it can be developed as a therapeutic agent to treat existing estrogen-dependent breast cancer or to prevent premalignant foci from developing into breast cancer. The antiestrotrophic activity of AFP was reported to be localized to a peptide consisting of amino acids 447-480, a 34-mer peptide termed P447. A series of parsings and substitutions of amino acids in the P447 sequence was intended to identify the shortest analog which retained antiestrotrophic activity. Peptides related to P447 were generated using solid phase peptide synthesis. Several shorter peptides, including an 8-mer called P472-2 (amino acids 472-479, peptide sequence EMTPVNPG), retained activity, whereas peptides shorter than eight amino acid residues were inactive. The dose-related antiestrotrophic activity of AFP-derived peptides was determined in an immature mouse uterine growth assay that measures their ability to inhibit estradiol-stimulated uterine growth. In this assay, the maximal inhibitory activities exhibited by peptide P472-2 (49%), by peptide P447 (45%), and by intact AFP (35-45%) were comparable. The octapeptide P472-2 was also active against estradiol-stimulated growth of T47D human breast cancer cells in culture. These data suggest that peptide P472-2 is the minimal sequence in AFP, which retains the antiestrotrophic activity found with the full-length molecule. The synthetic nature and defined structure of this 8-mer peptide suggest that it can be developed into a new drug which opposes the action of estrogen, perhaps including the promotional effects of estradiol in the development of human breast cancer.     

Structure Prediction for Peptides Capable of Inducing Antibody Formation in Mice

A simple method for the sequence prediction of peptides capable of thein vivo stimulation of antibody production in mice without conjugation with protein carriers was proposed on the basis of literature data on the structure of T-helper epitopes active in vivo. According to this approach, a potentially active peptide should contain a nine-membered sequence with a hydrophobic amino acid residue in the first position and a positively charged residue in the ninth position. The efficiency of this approach was confirmed by the presence of such sequences in the previously described synthetic peptides with immune activities, by the application of this approach to the choice of immunogenic fragments within the sequences of various proteins that exhibited further the specific activity, and by the construction of immunogenic peptides on the basis of inactive natural sequences.     

Folding pathway mediated by an intramolecular chaperone: propeptide release modulates activation precision of pro-subtilisin

Propeptides of several proteases directly catalyze the protein folding reaction. Uncatalyzed folding traps these proteases into inactive molten-globule-like conformers that switch into active enzymes only when their cognate propeptides are added in trans. Although tight binding and proteolytic susceptibility forces propeptides to function as single turnover catalysts, the significance of their inhibitory function and the mechanism of activation remain unclear. Using pro-subtilisin as a model, we establish that precursor activation is a highly coordinated process that involves synchronized folding, autoprocessing, propeptide release, and protease activation. Our results demonstrate that activation is controlled by release of the first free active protease molecule. This triggers an exponential cascade that selectively targets the inhibitory propeptide in the autoprocessed complex as its substrate. However, a mutant precursor that enhances propeptide release can drastically reduce the folding efficiency by altering the synergy between individual stages. Our results represent the first demonstration that propeptide release, not precursor folding, is the rate-determining step and provides the basis for the proposed model for precise spatial and temporal activation that allows proteases to function as regulators of biological function.     

Design and synthesis of novel antimicrobial peptides on the basis of alpha helical domain of Tenecin 1, an insect defensin protein, and structure-activity relationship study

Tenecin 1, a peptide consisting of 43 amino acids, exhibits a potent bactericidal activity against various Gram-positive bacteria and shares a common structural feature of insect defensin family corresponding to cysteine stabilized alpha/beta motif. Our previous research indicated that an active fragment was successfully extracted from C-terminal beta sheet domain of Tenecin 1, whereas the fragment corresponding to the alpha helical region of the protein had no antibacterial activity. We chose this inactive fragment corresponding to alpha helical region of Tenecin 1 and synthesized derivatives with a different net positive charge by using rational design. Interestingly, we successfully endowed antibacterial activity as well as antifungal activity to the inactive alpha helical fragment by single or double amino acid replacement(s) without an increase of hemolytic activity. The leakage of dye from vesicles induced by the active peptides suggested that these peptides act on the membranes of pathogen as a primary mode of action. Structure-activity relationship study of a series of the active derivatives revealed that amphiphilic structure and high net positive charge were prerequisite factors for the activity and that there was a relationship between the antibacterial activity and the isoelectric point of the active peptides. In this work, we showed an efficient method to endow the antibacterial activity as well as antifungal activity to the inactive fragment derived from a cyclic insect defensin protein and suggested a facile method to screen for active fragments from cyclic host defense peptides.     

类ACE抑制肽的合成及其活性分析

血管紧张素转换酶(angiotensin converting enzyme,ACE)通过作用于维持血压正常的肾素-血管紧张系统(rennin-angiotensin system, RAS)和激肽释放酶 激肽系统(kallikrein-kinin system, KKS),使其失衡导致血压升高．而ACE活性抑制肽可以竞争性地与ACE的活性中心结合,从而抑制ACE的活性,使血压降低．天然来源的ACE抑制肽与传统的降压药物相比效果较好,无毒副作用,对正常血压没有影响,对于高血压的治疗和人类健康具有重要意义. 本文以酪蛋白中提取的ACE活性抑制肽KVLPVP为先导肽,根据ACE抑制肽的结构特点,设计合成一系列的类ACE肽(similar ACE-like peptides). 利用反相高效液相色谱法(RP-HPLC)直接测定其体外ACE抑制活性. 结果表明,当芳香性的氨基酸残基Phe、Tyr、His和疏水性Val残基位于C-端时会提高多肽的ACE抑制活性,尤其是His位于C 端时,ACE抑制活性更强. 通过对比先导肽与所合成的类ACE肽的ACE活性抑制率,可以发现,类ACE肽的ACE活性抑制率均高于先导肽．基于不同氨基酸残基位于C-端时对多肽的ACE抑制活性的研究,可以为降血压药物分子设计和筛选提供基础.     

Isolation of a low molecular weight active fragment of potato proteinase inhibitor IIb.

A low molecular weight active fragment of potato proteinase inhibitor IIPB was obtained by incubating the inhibitor with an equimolar amount of trypsin [EC 3.4.21.4] at pH 8 and 30 degrees for 16 hr, followed by gel filtration through Sephadex G-50, treatment with trichloroacetic acid, and CM-cellulose chromatography. The purified active fragment consisted of a single peptide chain with a molecular weight of 4,300, comprising 39 amino acid residues. It retained very strong inhibitory activity against chymotrypsin [EC 3.4.21.1] and subtilisin [EC 3.4.21.14]. However, the yield of this active fragment was rather low and was variable. On further incubation with trypsin, it was converted into smaller inactive peptides.     

Characterization of delta-guaiene synthases from cultured cells of Aquilaria, responsible for the formation of the sesquiterpenes in agarwood

The resinous portions of Aquilaria plants, called agarwood, have been used as medicines and incenses. Agarwood contains a great variety of sesquiterpenes, and a study using cultured cells of Aquilaria showed the production of sesquiterpenes (α-guaiene, α-humulene, and δ-guaiene) to be induced by treatment with methyl jasmonate (MJ). In this study, the accumulation and production of sesquiterpenes were quantified. The amounts accumulated and produced reached a maximum at 12 h, and the most abundant product was α-humulene at 6 h and δ-guaiene after 12 h. However, a headspace analysis of the cells revealed that α-humulene is likely to be volatilized; so overall, the most abundant sesquiterpene in the cells was δ-guaiene. A cDNA library from RNA isolated from MJ-treated cells was screened using PCR methodologies to isolate five clones with very similar amino acid sequences. These clones were expressed in Escherichia coli, and enzymatic reactions using farnesyl pyrophosphate revealed that three of the clones yielded the same compounds as extracted from MJ-treated cells, the major product being δ-guaiene. These genes and their encoded enzymes are the first sesquiterpene synthases yielding guaiane-type sesquiterpenes as their major products to be reported. Expression of a fourth terpene synthase gene in bacteria resulted in the accumulation of the protein in insoluble forms. Site-directed mutagenesis of the inactive clone and three-dimensional homology modeling suggested that the structure of the N-terminal domain was important in facilitating proper folding of the protein to form a catalytically active structure.     

Conformation and lytic activity of eumenine mastoparan: a new antimicrobial peptide from wasp venom.

Eumenine mastoparan-AF (EMP-AF) is a novel membrane active tetradecapeptide recently isolated from the venom of solitary wasp, Anterhynchium flavomarginatum micado. It was reported previously that EMP-AF peptide presented low cytolytic activities in human erythrocytes and in RBL-2H3 mast cells. In the present work, we observed that this peptide is able to permeate anionic liposomes, and in less extension also the neutral ones. We present evidences showing that the permeation ability is well correlated with the amount of helical conformation assumed by the peptides in these environments. This peptide also showed a broad-spectrum inhibitory activity against Gram-positive and Gram-negative bacteria. The permeability of liposomes and the antibiotic effect showed a significant reduction when C-terminus was deamidated (in acidic form). The removal of the three first amino acid residues from the N-terminus rendered the peptide inactive both in liposomes and in bacteria. The results suggest that the mechanism of action involves a threshold in the accumulation of the peptide at level of cell membrane.     

Peptide inhibitors for angiotensin I-converting enzyme from thermolysin digest of dried bonito.

Dried bonito (Katsuobusi), a Japanese traditional seasoning made of bonito muscle was hydrolyzed by various proteases and the inhibitory activity of the hydrolyzates for angiotensin I-converting enzyme (ACE) [EC 3.4.15.1] was measured. Among the digests, thermolysin digest showed the most potent inhibitory activity. Eight inhibitory peptides were isolated from the digest using HPLC. The amino acid sequences of inhibitory peptides were Ile-Lys-Pro-Leu-Asn-Tyr, Ile-Val-Gly-Arg-Pro-Arg-His-Gln-Gly, Ile-Trp-His-His-Thr, Ala-Leu-Pro-His-Ala, Phe-Gln-Pro, Leu-Lys-Pro-Asn-Met, Ile-Tyr, and Asp-Tyr-Gly-Leu-Tyr-Pro. By searching for the sequence homology in many proteins, four of them were found in the primary structure of actin. Asp-Met-Ile-Pro-Ala-Gln-Lys was obtained from the boiling water extract of dried bonito and this peptide was found in the primary structure of creatine kinase. Fragments of these peptides were prepared by further enzymatic digestion or chemical synthesis and their ACE-inhibitory activities were measured. Among them, Ile-Lys-Pro, Ile-Trp, Leu-Lys-Pro, and Leu-Tyr-Pro had higher inhibitory activity than their parental peptides. Ile-Lys-Pro suppressed the hypertensive activity of angiotensin I.     

The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding

Recently, we documented that the short, proline-rich antibacterial peptides pyrrhocoricin, drosocin, and apidaecin interact with the bacterial heat shock protein DnaK, and peptide binding to DnaK can be correlated with antimicrobial activity. In the current report we studied the mechanism of action of these peptides and their binding sites to Escherichia coli DnaK. Biologically active pyrrhocoricin made of L-amino acids diminished the ATPase activity of recombinant DnaK. The inactive D-pyrrhocoricin analogue and the membrane-active antibacterial peptide cecropin A or magainin 2 failed to inhibit the DnaK-mediated phosphate release from adenosine 5'-triphosphate (ATP). The effect of pyrrhocoricin on DnaK's other significant biological function, the refolding of misfolded proteins, was studied by assaying the alkaline phosphatase and beta-galactosidase activity of live bacteria. Remarkably, both enzyme activities were reduced upon incubation with L-pyrrhocoricin or drosocin. D-Pyrrhocoricin, magainin 2, or buforin II, an antimicrobial peptide involved in binding to bacterial nucleic acids, had only negligible effect. According to fluorescence polarization and dot blot analysis of synthetic DnaK fragments and labeled pyrrhocoricin analogues, pyrrhocoricin bound with a K(d) of 50.8 microM to the hinge region around the C-terminal helices D and E, at the vicinity of amino acids 583 and 615. Pyrrhocoricin binding was not observed to the homologous DnaK fragment of Staphylococcus aureus, a pyrrhocoricin nonresponsive strain. In line with the lack of ATPase inhibition, drosocin binding appears to be slightly shifted toward the D helix. Our data suggest that drosocin and pyrrhocoricin binding prevents the frequent opening and closing of the multihelical lid over the peptide-binding pocket of DnaK, permanently closes the cavity, and inhibits chaperone-assisted protein folding. The biochemical results were strongly supported by molecular modeling of DnaK-pyrrhocoricin interactions. Due to the prominent sequence variations of procaryotic and eucaryotic DnaK molecules in the multihelical lid region, our findings pave the road for the design of strain-specific antibacterial peptides and peptidomimetics. Far-fetched applications of the species-specific inhibition of chaperone-assisted protein folding include the control of not only bacteria but also fungi, parasites, insects, and perhaps rodents.     

Role of the lipase-specific foldase of Burkholderia glumae as a steric chaperone

Most lipases of Gram-negative bacteria require a lipase-specific foldase (Lif) in order to fold in the periplasm into their active, protease-resistant conformation prior to their secretion. The periplasmic domain of the Lif (amino acids 44-353) of Burkholderia glumae was purified as a His-tagged protein, and its function in the folding of lipase was studied in vitro. Refolding of the denatured lipase into its active conformation was dependent on the presence of the Lif. Circular dichroism revealed that the lipase refolded in the absence of Lif into a form with a native-like conformation, which was more stable against heat-induced denaturation than the native form, but was enzymatically inactive. This form of the protein could be activated by adding Lif after several hours, which demonstrates that the function of this chaperone is to help lipase to overcome an energetic barrier in the productive folding pathway rather than to prevent it from entering a non-productive pathway. The Lif was shown to interact with the native lipase in protease-protection experiments as well as by affinity chromatography, consistent with a role of the Lif late in the folding process. These results demonstrate that the Lif functions in a way analogous to the propeptides of many bacterial proteases and indicate that the amino acid sequence of the lipase does not contain all the information required for the protein to adopt its three-dimensional structure.