Plant defense and antimicrobial peptides

Plants are constantly exposed to a large array of pathogenic organisms and the survival in these conditions demands quick defense responses which include the synthesis of defense peptides and proteins with antimicrobial properties. The main groups of antimicrobial peptides found in plants are thionins, defensins and lipid transfer proteins. They constitute interesting candidates to engineer disease resistance in plants.     

Ribosomal synthesis of nonstandard peptides.

It is well known that standard peptides, which comprise proteinogenic amino acids, can act as specific chemical probes to target proteins with high affinity. Despite this fact, a number of peptide drug leads have been abandoned because of their poor cell permeability and protease instability. On the other hand, nonstandard peptides isolated as natural products often exhibit remarkable pharmaco-behavior and stability in vivo. Although it is likely that numerous nonstandard therapeutic peptides capable of recognizing various targets could have been synthesized, enzymes for nonribosomal peptide syntheses are complex; therefore, it is difficult to engineer such modular enzymes to build nonstandard peptide libraries. Here we describe an emerging technology for the synthesis of nonstandard peptides that employs an integrated system of reconstituted cell-free translation and flexizymes. We summarize the historical background of this technology and discuss its current and future applications to the synthesis of nonstandard peptides and drug discovery.     

Inhibition of procollagen cell-free synthesis by amino-terminal extension peptides

Peptides prepared from the amino termini of pro alpha 1(I) and pro alpha 1(III) collagen chains inhibit the production of pro alpha 1(I) and pro alpha 2 by rat calvaria rna in a reticulocyte cell-free system. The synthesis of other proteins was not altered, suggesting a specific effect on collagen production. Various peptides from the helical region of the alpha 1(I) chain did not alter translation. These studies, taken together with earlier studies showing inhibition of collagen synthesis by cells in culture receiving the amino-terminal peptides, are consistent with a regulatory function in collagen synthesis for the amino-terminal peptides from procollagen.     

Bioorganic synthesis of end-capped anti-HIV peptides by simultaneous cyanocysteine-mediated cleavages of recombinant proteins

Bioorganic synthesis of N- and C-terminal end-capped peptides by two simultaneous S-cyanocysteine-mediated cleavages of recombinant proteins is described. This approach is demonstrated in the preparation of anti-HIV fusion inhibitory peptides.     

Functionalization of peptides and proteins by aldehyde or keto groups

The functionalization of peptides and proteins by aldehyde or keto groups has become the subject of intensive research since the discovery of the inhibition properties of peptide aldehydes and the advent of protein engineering. The first part of this review focuses upon the tremendous efforts devoted to the solid-phase synthesis of peptide aldehydes as protease inhibitors. The second part describes the utility of the aldehyde or keto functionalities for the site-specific modification of peptides or proteins.     

Human immune responses to synthetic peptides from the Epstein-Barr nuclear antigen

Humans infected with Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, develop antibodies against a nuclear antigen (EBNA) that is present in virally transformed B lymphocytes. The EBNA protein contains a unique glycine-alanine repeating sequence. We have synthesized peptides corresponding to various regions of the EBNA molecule within and near this sequence. Rabbit antibodies against the peptides within the sequence reacted directly with the EBNA protein, as detected by Western blotting. The sera of individuals with antibodies against Epstein-Barr virus contained abundant antibodies also reactive with one or several of the synthetic peptides within the sequence. Moreover, human antibodies against these simple peptides were induced specifically early in the course of infectious mononucleosis. When compared with normal controls, antibody levels to the glycine-alanine peptides were significantly higher in patients with rheumatoid arthritis and progressive systemic sclerosis, but not in patients with two other autoimmune diseases. These results document that i) antibodies against the peptides detect the EBNA protein, ii) humans infected with EBV produce high titers of antibodies reactive with these synthetic antigens, and iii) antibody titers against the peptides are abnormally elevated in certain autoimmune diseases.     

Synthesis of peptides as cloned ubiquitin extensions

Oligonucleotides encoding four peptides ranging in length from 10 to 21 amino acids were cloned between the Afl2 and KpnI sites in the ubiquitin expression vector, pNMHUb. Escherichia coli AR13, a strain that contains a temperature-sensitive lambda repressor, was transformed by the plasmids, and upon shift to 42 degrees C, the cells produced large amounts of ubiquitin extended at its carboxyl terminus by each of the four peptides. Following a simple three-step purification of the ubiquitin fusion proteins, the peptides were cleaved from ubiquitin using a ubiquitin-alpha-protein hydrolase isolated from rabbit reticulocytes. The released ubiquitin molecules were shown to be competent in conjugation assays, and amino acid analyses of the purified peptides revealed that full length products were obtained in all cases. Since peptide yields varied from 2 to 4 mg/liter of culture medium, ubiquitin extensions provide an attractive alternative to solid phase synthesis for the production of peptides.     

Synthesis and characterization of histidine-phosphorylated peptides.

Posttranslational phosphorylation of proteins is an important event in many cellular processes. Whereas phosphoesters of serine, threonine, and tyrosine have been studied extensively, only limited information is available for other amino acids modified by a phosphate group. The formation of phosphohistidine residues in proteins was discovered originally in prokaryotic organisms, but also has been found recently in eukaryotic cells. We describe methods for the synthesis and analysis of phosphohistidine-containing peptides, a prerequisite for the investigation of the role of this posttranslational modification in cellular processes.     

Inhibiting effect of procollagen peptides on collagen biosynthesis in fibroblast cultures.

NH2-terminal extension peptides of type I and type III procollagens were isolated from dermatosparactic and normal fetal calfskin, respectively. Cell culture experiments showed that the globular domains of the tested procollagen peptides were biologically active but that peptides from the helical region of collagen had no effect. The peptides were added to the incubation medium of calf fibroblasts along with radioactive precursor amino acids, and the amount of newly synthesized collagen was determined. The experiments indicated that procollagen peptides exerted a feedback-like inhibitory effect specific for the synthesis of collagen. Neither degradation of collagen, hydroxylation of collagen alpha chains, nor synthesis of noncollagenous proteins were affected. Synthesis of type II collagen by calf chondrocytes was not reduced. In addition, it was shown that procollagen peptides from calf were equally effective when added to human fibroblast cultures, an observation that could be of considerable medical interest.     

A family of bacteria-regulated, cecropin D-like peptides from Manduca sexta

Manduca sexta larvae respond to bacterial challenge by synthesizing a set of antibacterial hemolymph proteins. We have purified and sequenced three members of a family of cecropin D-like bactericidal peptides and isolated a cDNA clone complementary to a closely related bactericidin. Results obtained by Northern hybridization and RNase protection analysis showed that the increased synthesis of bactericidins is due to induction of their mRNA levels and that the synthesis of these peptides is not strictly tissue-specific, in contrast to previous beliefs. Although fat body was a richer source, the relative amounts of bactericidin mRNA were significant in seven other tissues examined.     

Isosteric replacement of sulfur with other chalcogens in peptides and proteins.

The review addresses the functional and structural properties of the two series of chalcogen analogues of amino acids in peptides and proteins, the methionine and the serine/cysteine series, and discusses the synthesis of the related selenium/tellurium analogues as well as their use in peptide synthesis and protein expression. Advances in synthetic methodologies and recombinant technologies and their combined applications in native and expressed protein ligation allows the isomorphous character of selenium- and tellurium-containing amino acids to be exploited for production of heavy metal mutants of proteins and thus to facilitate the phasing problem in x-ray crystallography. In addition, selenocysteine has been recognized as an ideal tool for the production of selenoenzymes with new catalytic activities. Moreover, the fully isomorphous character of disulfide replacement with diselenide is well suited to increase the robustness of cystine frameworks in cystine-rich peptides and proteins and for the de novo design of even non-native cystine frameworks by exploiting the highly negative redox potential of selenols.     

Optimizing synthesis and expression of transmembrane peptides and proteins

Structural studies of full-length membrane proteins have been hindered by their hydrophobicity and low expression in a variety of systems. However, a simplifying aspect of membrane protein folding is that individual transmembrane segments or membrane protein fragments have been observed to represent independent folding domains, and as such, can facilitate the study of packing interactions between TM helices, and the collection of structural information regarding membrane proteins. This review focuses on two categories of techniques--total peptide synthesis and bacterial expression--that can each be optimized for preparation of transmembrane protein segments. First, synthesis of hydrophobic transmembrane peptides that are N- and/or C-tagged with solubilizing residues such as lysine can improve manipulation of the transmembrane core in a variety of biophysical experiments. In this context, we describe general protocol considerations during the synthesis, cleavage, and purification stages of these peptides to identify appropriate parameters that combine to improve yields of hydrophobic peptides. Second, bacterial expression of membrane protein fragments is a useful tool for producing large quantities of hydrophobic protein segments. Targeting protein expression within Escherichia coli can facilitate purification, while attaching the hydrophobic construct to a hydrophilic fusion protein can amplify expression. We show that adapting protein constructs to comply with expression host specifications, in concert with thorough exploration of expression conditions such as the type of media used for expression, temperature, and cell strain, can significantly improve protein yields.     

Solid-phase synthesis of glucose-derived Amadori peptides.

Nonenzymatic glycosylation or glycation of amino groups in peptides and proteins by D-glucose is a universal reaction with important implications for the pathogenesis of many diseases including diabetes mellitus. Here a general approach is reported to synthesize site specifically glucose-derived N-glycated peptides. Therefore, model peptides H-AKASASFL-NH(2), H-AKASADFL-NH(2), H-ASKASKFL-NH(2), and H-AKDSASFL-NH(2) were synthesized on solid phase by Fmoc chemistry using Fmoc-Lys(4-methyltrityl)-OH in positions 2 or 3 to be glycated. After completion of the synthesis, the acid labile 4-methyltrityl-group was cleaved with 1% TFA in DCM and the free amino groups were glycated by the Lobry de Bruyn reaction using 2,3:4,5-di-O-isopropylidene-aldehydo-beta-D-arabino-hexos-2-ulo-2,6-pyranose on solid phase. After TFA treatment, the crude peptides were obtained in high yields and purities above 80%. Minor by-products were well separated on reversed-phase HPLC.     

Novel peptides with tyrosinase inhibitory activity

Tyrosinase inhibition by peptides may find its application in food, cosmetics or medicine. In order to identify novel tyrosinase inhibitory peptides, protein-based peptide libraries made by SPOT synthesis were used to screen for peptides that show direct interaction with tyrosinase. One of the peptide libraries studied consists of overlapping, octameric peptides derived from industrial proteins as beta-casein, alpha-lactalbumin, beta-lactoglobulin, ovalbumin, gliadin, glycinin, and beta-conglycinin. On-membrane activity staining resulted in a set of peptides that are not only able to bind to tyrosinase, but are able to inhibit tyrosinase as well. Peptides containing aspartic or glutamic acid residues usually do not bind very well to tyrosinase. Strong tyrosinase-binding peptides always contain one or more arginine residues, often in combination with phenylalanine, while lysine residues can be found equally among nonbinding peptides as well as moderate tyrosinase-binding peptides. The presence of the hydrophobic, aliphatic residues valine, alanine or leucine appears to be important for tyrosinase inhibition. Therefore, good tyrosinase inhibitory peptides preferably contain arginine and/or phenylalanine in combination with valine, alanine and/or leucine.     

Formation of disulfide bonds in proteins and peptides

For many proteins and peptides, disulfide bridges are prerequisite for their proper biological function. Many commercialized proteins are crosslinked by disulfide bridges that increase their resistance to destructive effects of extreme environment used in industrial processes or protect protein-based therapeutics from rapid proteolytic degradation. Manufacturing of these products must take into account oxidative refolding--a formation of native disulfide bonds by specific pairs of cysteines located throughout a sequence of linear protein. This review describes basic and practical aspects of oxidative folding that should be considered while designing and optimizing manufacturing of proteins using chemical synthesis, semi-synthesis and a recombinant expression.     

The role of antimicrobial peptides in innate immunity

Production of antimicrobial peptides and proteins is an importantmeans of host defense in eukaryotes. The larger antimicrobialproteins, containing more than 100 amino acids, are often lyticenzymes, nutrient-binding proteins or contain sites that targetspecific microbial macromolecules. The smaller antimicrobialpeptides act largely by disrupting the structure or functionof microbial cell membranes. Hundreds of antimicrobial peptideshave been found in the epithelial layers, phagocytic cells andbody fluids of multicellular animals, from mollusks to humans.Some antimicrobial peptides are produced constitutively, othersare induced in response to infection or inflammation. Studiesof the regulation of antimicrobial peptide synthesis in Drosophilahave been particularly fruitful, and have provided a new paradigmfor the analysis of mammalian host defense responses. It nowappears that the general patterns of antimicrobial responsesof invertebrates have been preserved in vertebrates ("innateimmunity") where they contribute to host defense both independentlyand in complex interplay with adaptive immunity.     

Food-derived peptides and intestinal functions

Many researchers have reported that food proteins and their peptides expressed a variety of functions in the body, including a reduction of blood pressure, modulation of immune cell functions, and regulation of nerve functions. However, food-derived proteins and peptides also play important roles in the intestinal tract before being absorbed. For example, some of the proteins and peptides can regulate the activity of digestive enzymes in the intestinal tract, thereby modulating the nutrient absorption in the intestines. These proteins and peptides have been used for functional foods with blood glucose- and blood cholesterol-lowering effects. Enhancement of the intestinal calcium absorption by casein-derived peptides is another example, such peptides being used as functional food ingredients. We have recently observed that certain milk peptides might stimulate the calcium transporter in intestinal epithelial cells. Carnosine, a dipeptide contained in skeletal muscles, was observed to suppress the secretion of inflammatory cytokines by intestinal epithelial cells that had been exposed to oxidative stress. Understanding the behavior of dietary proteins and peptides in the intestines is important for designing functional foods with physiological functions.     

The use of synthetic tRNAs as probes for examining nascent peptides on Escherichia coli ribosomes.

The polyuridylic acid-dependent syntheses of polycysteine and polyserine were carried out on Escherichia coli ribosomes using two new synthetic tRNA species. The peptides were initiated with N-acetyl or N-acyl coumarin derivatives of either Ser-tRNA or Phe-tRNA. The properties of the resulting nascent peptides were compared to those of nascent polyphenylalanine chains synthesized under similar conditions. This was accomplished by following changes in the fluorescence properties of the probes covalently linked to the amino-terminus of each of the nascent polypeptides as they were formed on the ribosomes. Nascent polycysteine and polyserine peptides appeared quite different from those of polyphenylalanine, as indicated by the anisotropy of fluorescence from the amino terminal probe. In contrast to serine and cysteine peptides, the synthesis of all the polyphenylalanine peptides was insensitive to inhibition by erythromycin, even though these peptides were initiated with N-acyl serine. The results support the hypothesis that nascent polyphenylalanine peptides have atypical physical and chemical properties and demonstrate the utility of using modified tRNAs to study ribosome function and the synthesis of proteins.     

The simultaneous synthesis of peptides and oligonucleotides on kaolinite with the participation of aminoacyladenylates

A simultaneous synthesis of peptides (2-5 residues) and oligonucleotides (3-9 residues) has been carried out on caolinite matrix using amino acids and aminoacyladenylates as substrates. The rate of oligomer synthesis on mineral surface is higher than that in solution. The mechanism of synthesis has been described. The data has been discussed in connection with abiogenesis of two major types of biopolymers, proteins and nucleic acids.