Peptide aptamers: exchange of the thioredoxin-A scaffold by alternative platform proteins and its influence on target protein binding

Peptide aptamers have emerged as powerful new tools for molecular medicine. They can specifically bind to and functionally inactivate a given target molecule under intracellular conditions. Typically, peptide aptamers are generated by screening a randomized peptide expression library, displayed from the Escherichia coli thioredoxin A (TrxA) protein. Here, we transferred peptide moieties from defined TrxA-based peptide aptamers to alternative scaffold proteins, such as the green fluorescent protein and staphylococcal nuclease. Yeast and mammalian two-hybrid assays as well as in vitro binding analyses show that the TrxA scaffold can be a major determinant for the binding of peptide aptamers. In addition, we demonstrate that TrxA can correctly display peptide sequences that correspond to the binding domains of natural interaction partners. Therefore, sequence analyses of TrxA-based peptide aptamers, isolated by two-hybrid screening from randomized expression libraries, should also be useful to find cellular binding partners for a given target protein, by homology. Received 1 August 2002; received after revision 17 September 2002; accepted 19 September 2002 RID="*" ID="*"Corresponding author.     

Release of calcitonin gene-related peptide from a human medullary carcinoma of the thyroid in vitro

The secretion of human calcitonin gene-related peptide was examined in perifusates of medullary carcinoma of the thyroid with a sensitive radioreceptor assay. Calcitonin gene-related peptide was released after the addition of calcium (25-100 mM), in a dose-dependent manner. The results indicate that human medullary carcinomas of the thyroid secrete the calcitonin gene-related peptide as well as calcitonin.     

Release of calcitonin gene-related peptide from a human medullary carcinoma of the thyroid in vitro

Summary The secretion of human calcitonin gene-related peptide was examined in perifusates of medullary carcinoma of the thyroid with a sensitive radioreceptor assay. Calcitonin gene-related peptide was released after the addition of calcium (25–100 mM), in a dose-dependent manner. The results indicate that human medullary carcinomas of the thyroid secrete the calcitonin gene-related peptide as well as calcitonin.     

Directed biosynthesis of analogues of ergot peptide alkaloids withClaviceps purpurea

Summary Analogues of peptide ergot alkaloids can be obtained by feeding a producing culture ofClaviceps purpurea with an analogue of one of the amino acids of the peptide chain of the alkaloid.     

Synthetic simulation of nonribosomal peptide biosynthesis. A dual role of alkylthiol esters in peptide synthesis

Summary Coupling of peptide alkylthiol esters with amino acid derivatives in the presence of pivalic acid or 2-hydroxypyridine proceeds without racemization. A dual role of alkylthiol esters as protective and reactive functions in peptide synthesis was well proved.     

The chemical structure of capreomycin.

The chemical structure of capreomycin, antituberculous peptide antibiotic, was revised from the results of NMR-analysis in comparison with tuberactinomycins. Capreomycin IA and IB were concluded to possess the similar amino acid sequences in their cyclic peptide moieties to those of tuberactinomycins.     

Structure and function of eukaryotic peptide transporters

The cotransport of protons and peptides is now recognised as a major route by which dietary nitrogen is absorbed from the intestine, and filtered protein reabsorbed in the kidney. Recently, molecular biology has had a very substantial impact on the study of peptide transport, and here we review the molecular and functional information available within the framework of physiology. To this end we consider not only the mammalian peptide transporters and their tissue distribution and regulation but also those from other species (including Caenorhabditis elegans) which make up the proton-dependent oligopeptide transport superfamily. In addition, understanding the binding requirements for transported substrates may allow future design and targeted tissue delivery of peptide and peptidomimetic drugs. Finally, we aim to highlight some of the less well understood areas of peptide transport, in the hope that it will stimulate further research into this challenging yet exciting topic.     

A new solid-phase synthesis of porcine vasoactive intestinal peptide using Nα-9-fluorenylmethyloxycarbonyl amino acids

Summary The solid-phase synthesis of an octacosapeptide amide corresponding to the amino acid sequence of porcine vasoactive intestinal peptide (VIP) is described. No final treatment with strong, anhydrous acid was employed, since the use of base-labile 9-fluorenylmethyloxycarbonyl amino acids bearing tert-butyl based side chain protection enabled the peptide chain assembly to be performed on p-benzyloxybenzyl amine resin, which was cleaved from the whole peptide amide at the end of the synthesis by diluted trifluoroacetic acid.     

The chemical structure of capreomycin

Summary The chemical structure of capreomycin, antituberculous peptide antibiotic, was revised from the results of NMR-analysis in comparison with tuberactinomycins. Capreomycin IA and IB were concluded to possess the similar amino acid sequences in their cyclic peptide moieties to those of tuberactionomycins.     

Comparative study of cysteine peptides of monomers and polymers of IgA]

The cysteinyl peptides of monomeric and polymeric IgA were studied by comparative autoradiography. Autoradiographies of polymeric IgA (containing or lacking J chain) showed an extra cysteinyl peptide which was never found in the monomeric forms. A similar peptide was obtained when reduced IgA was cleaved by BrCN. The possible identity of this fragment is discussed.     

Spleen antibacterial peptides: high levels of PR-39 and presence of two forms of NK-lysin

Antibacterial peptides were isolated from porcine spleen by acetic acid extraction, ion exchange chromatography and reverse-phase high-performance liquid chromatography. C-terminal ladder sequence analysis of a bioactive peptide with matrix-assisted laser desorption/ionization mass spectrometry after digestion with carboxypeptidases P and Y showed that it is identical to the antibacterial proline/arginine-rich intestinal peptide PR-39. It is present at high levels in granulocytes of the spleen, and peptides with C-terminal proline amide and internal adjacent Pro residues can be analyzed with this method. In addition, two forms of NK-lysin (NKL) were found. One, NKLi, is identical to that isolated from pig intestine, and the other, NKLbw, to a mature peptide deduced from a clone from a porcine bone marrow cDNA library.     

Regulatory peptide receptors: visualization by autoradiography

The receptors for regulatory peptides have been extensively characterized using radioligand binding techniques. By combining these binding techniques with autoradiography it is possible to visualize at the light and electron microscopic levels the anatomical and cellular localization of these receptors. In this review we discuss the procedures used to label peptide receptors for autoradiography and the peculiarities of peptides as ligands. The utilization of autoradiography in mapping peptide receptors in brain and peripheral tissues, some of the new insights revealed by these studies particularly the problem of 'mismatch' between endogenous peptides and receptors, the existence of multiple receptors for a given peptide family and the use of peptide receptor autoradiography in human tissues are also reviewed.     

Peptide synthesis through evolution

Ribosome-catalyzed peptide bond formation is a crucial function of all organisms. The ribosome is a ribonucleoprotein particle, with both RNA and protein components necessary for the various steps leading to protein biosynthesis. Evolutionary theory predicts an early environment devoid of complex biomolecules, and prebiotic peptide synthesis would have started in a simple way. A fundamental question regarding peptide synthesis is how the current ribosome-catalyzed reaction evolved from a primitive system. Here we look at both prebiotic and modern mechanisms of peptide bond formation and discuss recent experiments that aim to connect these activities. In particular, RNA can facilitate peptide bond formation by providing a template for activated amino acids to react and can catalyze a variety of functions that would have been necessary in a pre-protein world. Therefore, RNA may have facilitated the emergence of the current protein world from an RNA or even prebiotic world.Received 4 December 2003; received after revision 13 January 2004; accepted 15 January 2004     

New approaches to therapy of cancers of the stomach,colon and pancreas based on peptide analogs

Cancers of the stomach, colon and exocrine pancreas are major international health problems and result in more than a million deaths worldwide each year. The therapies for these malignancies must be improved. The effects of gastrointestinal (GI) hormonal peptides and endogenous growth factors on these cancers were reviewed. Some GI peptides, including gastrin and gastrin-releasing peptide (GRP) (mammalian bombesin), appear to be involved in the growth of neoplasms of the GI tract. Certain growth factors such as insulin-like growth factor (IGF)-I, IGF-II and epidermal growth factor and their receptors that regulate cell proliferation are also implicated in the development and progression of GI cancers. Experimental investigations on gastric, colorectal and pancreatic cancers with analogs of somatostatin, antagonists of bombesin/GRP, antagonists of growth hormone-releasing hormone as well as cytotoxic peptides that can be targeted to peptide receptors on tumors were summarized. Clinical trials on peptide analogs in patients with gastric, colorectal and pancreatic cancers were reviewed and analyzed. It may be possible to develop new approaches to hormonal therapy of GI malignancies based on various peptide analogs.Received 20 November 2003; accepted 6 January 2004     

A molecular recognition hypothesis for nonpeptides: Na+ K+ ATPase and endogenous digitalis-like peptides

The molecular recognition hypothesis for peptides is that binding sites of ligands and their receptors are encoded by short, complementary segments of DNA. A corollary hypothesis for nonpeptide ligands posited here is that peptide replicas may be encoded by the DNA segment complementary to the receptor binding sites for nonpeptides. This corollary was tested for digitalis, a family of cardiotonic and natriuretic steroids including ouabain. A hexapeptide (ouabain-like peptide, OLP) complementary to a ouabain binding site on sodium/potassium dependent adenosine triphosphatase (Na⁺ K⁺ ATPase) exhibited activity in a digitalis bioassay. Antisera to the complementary peptide (OLP) stained the neurohypophysis in an immunocytochemical procedure. The complementary peptide was found to share an identical 4-amino acid region with the 39-amino acid glycopeptide moiety of the vasopressin-neurophysin precursor. This glycopeptide was isolated from pituitary extracts; it exhibited digitalis-like activity in the submicromolar range and cross-reacted with complementary peptide antibodies. Another digitalis-like substance with high activity also was detected in the extracts. These results demonstrate that the vasopressin-neurophysin glycopeptide has digitalis-like activity. Moreover, the findings are consistent with the hypothesis that peptide mimetics of nonpeptides are encoded in the genome. Received 23 November 1998; received after revision 18 January 1999; accepted 19 February 1999     

Structural differences between somatic H2B and testis-specific TH2B histones of the rat

Summary Testis-specific histone TH2B from rat testis showed a very similar tryptic peptide map to somatic H2B from rat liver, indicating a common evolutionary origin. However, 5 peptide differences were detected between the 2 proteins, and the amino acid compositions of these distinctive peptides were determined.Supported by Grant 780-0609 from the Ford Foundation.     

Pharmacologically active spider peptide toxins

Advances in mass spectrometry and peptide biochemistry coupled to modern methods in electrophysiology have permitted the isolation and identification of numerous novel peptide toxins from animal venoms in recent years. These advances have also opened up the field of spider venom research, previously unexplored due to methodological limitations. Many peptide toxins from spider venoms share structural features, amino acid composition and consensus sequences that allow them to interact with related classes of cellular receptors. They have become increasingly useful agents for the study of voltage-sensitive and ligand-gated ion channels and the discrimination of their cellular subtypes. Spider peptide toxins have also been recognized as useful agents for their antimicrobial properties and the study of pore formation in cell membranes. Spider peptide toxins with nanomolar affinities for their receptors are thus promising pharmacological tools for understanding the physiological role of ion channels and as leads for the development of novel therapeutic agents and strategies for ion channel-related diseases. Their high insecticidal potency can also make them useful probes for the discovery of novel insecticide targets in the insect nervous system or for the development of genetically engineered microbial pesticides.Received 19 March 2003; received after revision 9 May 2003; accepted 16 May 2003     

Chemical synthesis of a hexadecapeptide segment of ubiquitin that activates adenylate cyclase and induces lymphocytes to differentiate

Summary A hexadecapeptide corresponding to positions 59–74 of ubiquitin was synthesized and purified. The peptide was characterized by its mobility in TLC and electrophoresis, amino acid sequence and composition, and molar rotation. The peptide possessed approximately 40% activity compared with native ubiquitin in each of 3 biological assays in vitro: a) thymocyte induction, b) B cell induction and c) elevation of intracellular cyclic AMP levels in sarcoma 180 cells.Acknowledgments. We thank Dr Geoffrey Tregear for helpful suggestions for the synthesis strategy, Dr Jim Burton for guidance with the methods for establishing peptide purity, and Ronald King, Miriam Miller, Jeanette Dilley and Linda Townley for excellent technical assistance. This work was supported by U.S. Public Health Service Grants CA-08748, CA-16889, CA-08415, CA-17085 and AI-12487, and by NCI Contract CB-53868.     

Galanin – 25 years with a multitalented neuropeptide

The skin, the largest organ of the body, functions as a barrier between the body proper and the external environment, as it is constantly exposed to noxious stressors. During the last few years, the concept of an interactive network involving cutaneous nerves, the neuroendocrine axis, and the immune system has emerged. The neuroendocrine system of the skin is composed of locally produced neuroendocrine mediators that interact with specific receptors. Among these mediators are neuropeptides, including members of the galanin peptide family--galanin, galanin-message-associated peptide, galanin-like peptide, and alarin--which are produced in neuronal as well as nonneuronal cells in the skin. Here we review the expression of the galanin peptides and their receptors in the skin, and the known functions of galanin peptides in different compartments of the skin. We discuss these data in light of the role of the galanin peptide family in inflammation and cell proliferation.     

Regulatory peptides in the respiratory system

Many regulatory peptides have been described in the respiratory tract of animals and humans. Some peptides (bombesin, calcitonin, calcitonin gene-related peptide) are localised to neuroendocrine cells and may have a trophic or transmitter role. Others are localised to motor nerves. Vasoactive intestinal peptide and peptide histidine isoleucine are candidates for neurotransmitters of non-adrenergic inhibitory fibres and may be cotransmitters in cholinergic nerves. These peptides may regulate airway smooth muscle tone, bronchial blood flow and airway secretions. Sensory neuropeptides (substance P, neurokinin A and B, calcitonin gene-related peptide) may contract airway smooth muscle, stimulate mucus secretion and regulate bronchial blood flow and microvascular permeability. If released by an axon reflex mechanism these peptides may be involved in the pathogenesis of asthma. Other peptides, such as galanin and neuropeptide Y, are also present but their function is not yet known.