Correlation between the compact regions predicted by the Average Distance Map (ADM) and the biologically active sites in atrial natriuretic peptide

The prediction of the short-range compact regions of human atrial natriuretic peptide (a-hANP), one of the biologically active peptides, has been made by means of the Average Distance Map(ADM). We found out that the location of the predicted short-range compact regions is consistent with the structural units determined by the NMR analysis (Kobayashiet al., 1988). Furthermore, the short-range compact regions correspond well to the biologically active areas of atriopeptin (103–125)-amide (which is homologous peptide toa-hANP), detected by the glycine substitution technique (Konishiet al., 1987). The results suggest that a predicted short-range compact region can be regarded as a possible active site in a biologically active peptide.     

Discrimination of folding types of globular proteins based on average distance maps constructed from their sequences

It has been shown that probable portions which form contacts in a protein can be predicted by means of an average distance map (ADM) as well as regular structures (-helices and -turns) defined as short-range compact regions (Kikuchiet al., 1988a,c). In this paper, we analyze the occurrence of those portions and short-range compact regions on ADMs for various proteins regarding their folding types. We have found out that each folding type of proteins shows characteristic distribution of such parts on ADMS. We also discuss the possibility of the prediction of folding types of proteins by ADMs.     

Prediction of probable pathways of folding in globular proteins

A method is described for the prediction of probable folding pathways of globular proteins, based on the analysis of distance maps. It is applicable to proteins of unknown spatial structure but known amino acid sequence as well as to proteins of known structure. It is based on an objective procedure for the determination of the boundary of compact regions that contain high densities of interresidue contacts on the distance map of a globular protein. The procedure can be used both with contact maps derived from a known three-dimensional protein structure and with predicted contact maps computed by means of a statistical procedure from the amino acid sequence alone. The computed contact map can also be used to predict the location of compact short-range structures, viz. -helices and -turns, thereby complementing other statistical predictive procedures. The method provides an objective basis for the derivation of a theoretically predicted pathway of protein folding, proposed by us earlier [Tanaka and Scheraga (1977) Macromolecules10, 291–304; Némethy and Scheraga (1979) Proc. Natl. Acad. Sci., U.S.A.76, 6050–6054].     

Large-scale Identification of Endogenous Secretory Peptides Using Electron Transfer Dissociation Mass Spectrometry

Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554–577]-NH₂, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics.Biologically active peptides, commonly known as peptide hormones and antimicrobial peptides, belong to a defined set of endogenous peptides that gain specialized functions not ascribed to original precursor proteins. For a precursor protein to generate such peptides, it must undergo specific cleavages and in some cases needs to be modified at specific sites (1). This limited cleavage, or proteolytic processing, represents an important cellular mechanism by which molecular diversity of proteins is increased at the post-translational level. In the postgenome era, it is being recognized that localization of processing sites in secretory proteins facilitates the identification of biologically active peptides. A standard approach to determining such sites is to use a panel of antibodies directed against different regions of a target protein (2). However, it is practically impossible to prepare antibodies that can thoroughly cover potential processing products arising from the precursor. Alternatively, mass spectrometry-assisted unbiased analysis of endogenous peptides may be a major step toward elucidating proteolytic processing (3).In neurons and endocrine cells, a majority of biologically active peptides are released via the regulated secretory pathway. They are stored in secretory granules and await secretion until the cells receive an exocytotic stimulus. Owing to their compartmentalization, secretory peptides can be noninvasively recovered in culture supernatant. We have shown that a data set of endogenous peptide sequences that are collected by this procedure is applicable to infer processing sites, as well as to identify bona fide processing products (4). Rather than being digested, every endogenous peptide should be analyzed in its native form to understand how the peptide is generated and subsequently degraded. However, it remains a challenge to identify endogenous peptides because of size heterogeneity (ranging from 3 aa to 100 aa). For example, thyrotropin-releasing hormone is a small 3-aa peptide, human adrenomedullin occurs as a 52-aa peptide, and a 98-aa N-terminal propeptide from the atrial natriuretic peptide precursor is found in the circulation. Unlike digested protein fragments used in bottom-up proteomics, C termini of these endogenous peptides are not restricted to specific residues. Furthermore, proteolytic processing leads to the production of peptides containing multiple internal basic residues, for which collision induced dissociation (CID)¹ shows limited performance (5).A solution to address this issue in endogenous peptide sequencing might be the use of electron transfer dissociation (ETD) tandem mass spectrometry, which has been shown to provide a more complete series of fragment ions and hence a more confident sequence identification, along with the ability to leave labile post-translational modifications intact (6–10). The benefit of ETD in bottom-up proteomics has been increasingly documented, whereas endogenous peptides remain largely unexplored by ETD, despite the expectation that ETD would improve sequencing for larger peptides. In the few studies on endogenous peptides (11, 12), ETD did not cover large peptides exceeding 5000 Da. Because we have used CID to facilitate the discovery of previously unknown biologically active peptides (3, 13, 14), we were interested to see if ETD would be helpful to identify endogenous peptides that have escaped identification by CID. Here we conducted a large-scale identification of endogenous secretory peptides, ranging from 1000 to 15000 Da, using CID and ETD. We describe the merits of using ETD, in connection with CID, in peptidomics studies. The most significant finding is the identification of a previously unknown peptide, VGF[554–577]-NH₂, which was sequenced solely by ETD. This peptide was found to have antimicrobial activity.     

Snapshot Peptidomics of the Regulated Secretory Pathway

Neurons and endocrine cells have the regulated secretory pathway (RSP) in which precursor proteins undergo proteolytic processing by prohormone convertase (PC) 1/3 or 2 to generate bioactive peptides. Although motifs for PC-mediated processing have been described ((R/K)X_n(R/K) where n = 0, 2, 4, or 6), actual processing sites cannot be predicted from amino acid sequences alone. We hypothesized that discovery of bioactive peptides would be facilitated by experimentally identifying signal peptide cleavage sites and processing sites. However, in vivo and in vitro peptide degradation, which is widely recognized in peptidomics, often hampers processing site determination. To obtain sequence information about peptides generated in the RSP on a large scale, we applied a brief exocytotic stimulus (2 min) to cultured endocrine cells and analyzed peptides released into supernatant using LC-MSMS. Of note, 387 of the 400 identified peptides arose from 19 precursor proteins known to be processed in the RSP, including nine peptide hormone and neuropeptide precursors, seven granin-like proteins, and three processing enzymes (PC1/3, PC2, and peptidyl-glycine α-amidating monooxygenase). In total, 373 peptides were informative enough to predict processing sites in that they have signal sequence cleavage sites, PC consensus sites, or monobasic cleavage sites. Several monobasic cleavage sites identified here were previously proved to be generated by PCs. Thus, our approach helps to predict processing sites of RSP precursor proteins and will expedite the identification of unknown bioactive peptides hidden in precursor sequences.The generation of peptide hormones or neuropeptides involves the proteolytic processing of precursor proteins by specific proteases. In neurons and endocrine cells, most, if not all, of these bioactive peptides are generated within the RSP¹ in which the processing enzymes PC1/3 or PC2 cleave precursors at basic residues (1, 2). The PC-mediated cleavage most often occurs at consecutive basic residues, but not all basic residues serve as PC recognition sites (2). This is partly because the secondary structure of a precursor also affects the substrate recognition (3). Identification of processing sites is hence a prerequisite for locating unknown peptides hidden in a precursor sequence.Peptidomics has been advocated to comprehensively study peptides cleaved off from precursor proteins by endogenous proteases (4–6). These naturally occurring peptides are beyond the reach of current proteomics and should be analyzed in their native forms. Unlike proteomics, peptidomics has the potential to uncover processing sites of precursor proteins. Most peptidomics studies, which target tissue peptidomes from brain or endocrine organs (7–11), have provided limited information about secretory peptides that could help to identify processing sites; they are too often blurred by subsequent actions of exopeptidases (cutting off a single amino acid or dipeptide from either end of a peptide).In MS-based identification of bioactive peptides present in biological samples, their relative low abundance in a total pool of naturally occurring peptides should be considered. Once extracted from cultured cells or tissues, bona fide secretory peptides and nonsecretory peptides or peptide fragments caused by degradation of abundant cytosolic proteins cannot be discriminated, and therefore we need to analyze samples rich in secretory peptides to facilitate the identification of bioactive peptides. Several attempts have been made to isolate secretory proteins or peptides, such as subcellular fractionation for harvesting secretory granules (12, 13). With all these efforts, a limited number of secretory peptides have been identified, and many known bioactive peptides still escape analysis.We took advantage of the fact that peptides processed in the RSP are enriched in secretory granules of neurons and endocrine cells and released on exocytosis. Here we applied a brief exocytotic stimulus (2 min) to cultured human endocrine cells and identified peptides released into supernatant using LC-MSMS on an LTQ-Orbitrap mass spectrometer. Nearly 97% of the identified peptides arose from precursor proteins known to be recruited to the RSP, such as peptide hormone precursors and granin-like secretory proteins. Our approach was validated by the identification of previously known processing sites of peptide hormone precursors. In addition, a majority of the identified peptides retained cleavage sites that agree with consensus cleavage sites for PCs, which are informative enough to deduce the processing sites of RSP proteins. This peptidomics approach will expedite the identification of unknown bioactive peptides.     

Conformational Properties of the CalliFMRF-Amide Series Neuropeptides

Conformational properties of five neuropeptides belonging to the calliFMRF-amide series with the Xaa-Pro-Yaa-Gln-Asp-Phe-Met-Arg-Phe-NH₂ homologous sequences were studied by the method of theoretical conformational analysis. Three members of these group [(1) (Xaa = Thr, Yaa = Gln), (2) (Xaa = Thr, Yaa = Ser), and (3) (Xaa = Yaa = Ser)] can stimulate the saliva secretion from the separated salivary gland of the Calliphora vomitoria fly, whereas two other calliFMRF-amides [(4) (Xaa = Lys, Yaa = Asn) and (5) (Xaa = Ala, Yaa = Gly)] are inactive in this biological test. Low-energy spatial structures of the studied compounds were determined by a conformational analysis. A comparison of the stable structures of the biologically active and inactive neuropeptides revealed a similarity in their conformational properties and allowed determination of the role of separate residues in the peptide folding. The calculations demonstrated that the C-terminal hexapeptide fragment identical in all the five peptides tends to form -helical structure, whereas the variable N-terminal tripeptide regions of calliFMRF-amides (1)–(5) form more conformationally flexible structures.     

Hypo-osmotic stimulation of active Na+ transport in frog muscle: Apparent upregulation of Na+ pumps

Summary The purpose of this work was to determine if hypotonicity, in addition to the stimulation of active Na⁺ transport (Venosa, R.A., 1978,Biochim. Biophys. Acta 510:378–383), promoted changes in (i) active K⁺ influx, (ii) passive Na^– and K⁺ fluxes, and (iii) the number of³H-ouabain binding sites.The results indicate that a reduction of external osmotic pressure () to one-half of its normal value (=0.5) produced the following effects: (i) an increase in active K⁺ influx on the order of 160%, (ii) a 20% reduction in Na⁺ influx and K⁺ permeability (P _K), and (iii) a 40% increase in the apparent density of ouabain binding sites. These data suggest that the hypotonic stimulation of the Na⁺ pump is not caused by an increased leak of either Na⁺ (inward) or K⁺ (outward). It is unlikely that the stimulation of active Na⁺ extrusion and the rise in the apparent number of pump sites produced by hypotonicity were due to a reduction of the intracellular ionic strength. It appears that, at least in part, the stimulation of active Na⁺ transport takes place whenever muscles are transferred from one medium to another of lower tonicity even if neither one was hypotonic (for instance =2 to =1 transfer). Comparison of the present results with those previously reported indicate that in addition to the number of pump sites, the cycling rate of the pump is increased by hypotonicity. Active Na⁺ and K⁺ fluxes were not significantly altered by hypertonicity (=2).     

Correlation of the conformation of a modified ribonuclease octapeptide, homologous to peptide T, with its ability to induce CD4-dependent monocyte chemotaxis

Peptide T, from the human immunodeficiency virus (HIV), whose sequence is Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr, has been shown to inhibit attachment of this virus to T cells and neural cells bearing the CD4 receptor. This peptide shares extensive homology with the 19–26 segment of ribonuclease A (RNase A), whose sequence is Ala-Ala-Ser-Ser-Ser-Asn-Tyr-Cys. Based on comparison of the structures of peptides occurring in proteins of known structure that are homologous to peptide T,viz, RNase A and endothiapepsin and on conformational energy calculations, we predicted that peptide T adopts a structure much like that for residues 19–26 in RNase A. A critical feature is a bend involving residues Thr 4-Asn 7 in peptide T corresponding to Ser 22-Tyr 25 in the RNase A peptide. Our proposed structure for peptide T has recently been confirmed by Cotelleet al. (Biochem. Biophys. Res. Commun. 171, 596–602). We now show directly that the RNase A peptide, with Met replacing Cys 26 to prevent disulfide exchange reactions, strongly induces monocyte-chemotaxis that is blocked by anti-CD4 monoclonal antibody. Both peptide T and RNase A fail to induce chemotaxis, however, in neutrophils which do not express surface CD4 receptors. These results suggest that both peptides interact with the CD4 receptor in inducing monocyte chemotaxis. We have also prepared cyclo-RNase A peptide with Met 26. Using molecular dynamics and conformational energy calculations, we find that the cyclic peptide cannot form a bend structure involving Ser 22-Tyr 25 that is superimposable on the RNase A bend. Indeed, we find that this peptide is inactive in inducing monocyte chemotaxis despite the fact that its amino acid sequence is identical to that of the open chain form. This result suggests that a correlation between the -bend structure of the RNase A peptide and peptide T and their abilities to bind to the CD4 receptor.     

Measurement of intrinsic exchange rates of amide protons in a 15N-labeled peptide

Summary We have used a modified version of a previously proposed technique, MEXICO [Gemmecker et al. (1993) J. Am. Chem. Soc., 115, 11620], and improved data analysis procedures in order to measure rapid hydrogen exchange (HX) rates of amide protons in peptides labeled only with ¹⁵N. The requirement of ¹³C-/¹⁵N-labeled material has been circumvented by adjusting conditions so that NOE effects associated with amide protons can be neglected (i.e., _0c~1). The technique was applied to an unstructured ¹⁵N-labeled 12-residue peptide to measure intrinsic HX rates, which are the essential reference for examining protein and peptide structure and dynamics through deceleration of HX rates. The method provided accurate HX rates from 0.5 to 50 s^-1 under the conditions used. The measured rates were in good agreement with those predicted using correction factors determined by Englander and co-workers [Bai et al. (1993) Proteins, 17, 75], with the largest deviations from the predicted rates found for residues close to the N-terminus. The exchange rates were found to exhibit significant sensitivity to the concentration of salt in the sample.     

Comparative aspects of natriuretic peptide physiology in non-mammalian vertebrates: a review

The natriuretic peptide system is a complex family of peptides and receptors that is primarily linked to the maintenance of osmotic and cardiovascular homeostasis. A natriuretic peptide system is present in each vertebrate class but there are varying degrees of complexity in the system. In agnathans and chondrichthyians, only one natriuretic peptide has been identified, while new data has revealed that multiple types of natriuretic peptides are present in bony fish. However, it seems in tetrapods that there has been a reduction in the number of natriuretic peptide genes, such that only three natriuretic peptides are present in mammals. The peptides act via a family of guanylyl cyclase receptors to generate the second messenger cGMP, which mediates a range of physiological effects at key targets such as the gills, kidney and the cardiovascular system. This review summarises the current knowledge of the natriuretic peptide system in non-mammalian vertebrates and discusses the physiological actions of the peptides.Abbreviations ANP atrial natriuretic peptide - AVT arginine vasotocin - BNP brain natriuretic peptide - cDNA complementary deoxyribonucleic acid - CNP C-type natriuretic peptide - cAMP adenosine 3, 5 cyclic monophosphate - cGMP guanosine 3, 5 cyclic monophosphate - GC guanylyl cyclase - GFR glomerular filtration rate - mRNA messenger ribonucleic acid - NPR natriuretic peptide receptor - NPs natriuretic peptides - sCP salmon cardiac peptide - VIP vasoactive intestinal peptide - VNP ventricular natriuretic peptide Communicated by I.D. Hume     

Amino acid sequence of the active site of human pseudocholinesterase

The usualE ₁ ^u and atypicalE ₁ ^a human pseudocholinesterases (acylocholine acylhydrolase, EC 3.1.1.8) were purified to homogeneity. The active-site serine residue was conjugated with diisopropyl fluorophosphate and digested with trypsin. The tryptic peptide containing the active site was isolated by gel filtration followed by two-dimensional paper chromatography and electrophoresis. The amino acid sequence of the active site peptide obtained from the usualE ₁ ^u enzyme was found to be Gly-Glu-Ser-Ala-Gly-Ala-Ser-Ala-Val-Ser-Leu. A remarkable structural homology exists between the human and the horse enzymes in their active sites. From the difference in electrophoretic mobility of the active-site peptides obtained from the usual and atypical enzymes, the probable structure of the atypical human enzyme was deduced as Gly-His-Ser-Ala-Gly-Ala-Ser-Ala-Val-Ser-Leu.     

Abundance-based Classifier for the Prediction of Mass Spectrometric Peptide Detectability Upon Enrichment (PPA)

The function of a large percentage of proteins is modulated by post-translational modifications (PTMs). Currently, mass spectrometry (MS) is the only proteome-wide technology that can identify PTMs. Unfortunately, the inability to detect a PTM by MS is not proof that the modification is not present. The detectability of peptides varies significantly making MS potentially blind to a large fraction of peptides. Learning from published algorithms that generally focus on predicting the most detectable peptides we developed a tool that incorporates protein abundance into the peptide prediction algorithm with the aim to determine the detectability of every peptide within a protein. We tested our tool, “Peptide Prediction with Abundance” (PPA), on in-house acquired as well as published data sets from other groups acquired on different instrument platforms. Incorporation of protein abundance into the prediction allows us to assess not only the detectability of all peptides but also whether a peptide of interest is likely to become detectable upon enrichment. We validated the ability of our tool to predict changes in protein detectability with a dilution series of 31 purified proteins at several different concentrations. PPA predicted the concentration dependent peptide detectability in 78% of the cases correctly, demonstrating its utility for predicting the protein enrichment needed to observe a peptide of interest in targeted experiments. This is especially important in the analysis of PTMs. PPA is available as a web-based or executable package that can work with generally applicable defaults or retrained from a pilot MS data set.Post-translational modification (PTM)¹ of proteins is a key regulatory mechanism in the vast majority of biological processes. Historically, to follow PTMs, site-specific antibodies had to be generated in a time-consuming and laborious process associated with high failure rates. Mass spectrometry (MS) holds enormous promise in PTM analysis as it is currently the only technique that has the ability to both discover, localize, and quantify proteome-wide modifications (1). Recent advances in instrumentation and method optimization makes it possible to detect the complete yeast proteome within one hour (2), an ever increasing proportion of the human proteome (3–6), and more than 10,000 phosphorylation sites in a single MS experiment (7, 8). As a result one of the major publicly available databases (www.phosphosite.org (9)) has curated >200,000 phosphorylation sites.Although the number of proteins and PTMs that can be identified is impressive, many modifications have still not been identified in any MS-based experiment. The identification and quantification of biologically relevant modifications is challenging for three reasons: (1) many proteins of interest are of very low abundance rendering them difficult to detect and quantify; (2) many modifications sites are present at substoichiometric quantities, further reducing their detectability; and (3) as large scale proteomics is based on the detection of peptides after a proteolytic digest, and the detectability of a peptide is determined by its physiochemical properties (10), many peptides from highly abundant proteins are never detected. This is particularly important, as there is a shift in the use of MS-based proteomics from large scale, unbiased, discovery-focused experiments toward directed experiments for accurate and precise quantification of biologically relevant PTMs. Protein and peptide enrichment strategies and/or targeted MS experiments like single reaction monitoring (SRM) (11) have increased the number of detectable peptides; however, both of these methods are laborious, and often not successful, that is, the peptide carrying the modification of interest is still not observed as it is fundamentally very difficult to detect.Protein enrichment is the method choice for most experimentalists, but there is no current way to determine whether this is likely to succeed prior to engaging in lengthy biochemical and/or analytical experiments. In an effort to gauge the chances of success for detecting a particular peptide we sought to develop an algorithm that can predict both the chances of detecting a particular peptide and, more importantly, what enrichment it would take to detect a particular peptide that is not easily detected. Here we present such a tool that predicts the detectability and estimates an enrichment factor, i.e. an increase in signal over the background that is necessary to actually detect a particular peptide. Our algorithm development was motivated by two premises: (1) In silico methods have been developed that focus on the prediction of easily detectable “proteotypic” peptides (peptides that are likely to provide the best detection sensitivity) with good accuracy (12–15). (2) Comprehensive proteome studies have shown that the number of detected peptides per protein, and thus the sequence coverage, varies with protein abundance (which is the basis for spectral counting-based protein quantification (16, 17)). We find that incorporation of protein abundance in a peptide classification tool improves the accuracy of the prediction of peptide detectability allowing us to predict the detectability of all peptides within a protein as well as the amount of enrichment needed to detect a peptide of interest.We used a set of 120 purified in vitro expressed proteins as a training set to develop a prediction tool. We deliver this in the form of a web-based interface that provides information about: (1) the probability of detecting the different tryptic peptides of a protein, and (2) the fold enrichment that would be required to bring a peptide of interest into the detectable range. This tool will help guide researchers in their efforts to monitor particular peptides and their modified cognates by MS, specifically, in prioritizing their efforts toward enriching proteins where they would be likely to be able to detect a peptide or modification of interest.     

Design,structural and functional characterization of a Temporin-1b analog active against Gram-negative bacteria

Background

Temporins are small antimicrobial peptides secreted by the Rana temporaria showing mainly activity against Gram-positive bacteria. However, different members of the temporin family, such as Temporin B, act in synergy also against Gram-negative bacteria. With the aim to develop a peptide with a wide spectrum of antimicrobial activity we designed and analyzed a series of Temporin B analogs.

Methods

Peptides were initially obtained by Ala scanning on Temporin B sequence; antimicrobial activity tests allowed to identify the TB_G6A sequence, which was further optimized by increasing the peptide positive charge (TB_KKG6A). Interactions of this active peptide with the LPS of E. coli were investigated by CD, fluorescence and NMR.

Results

TB_KKG6A is active against Gram-positive and Gram-negative bacteria at low concentrations. The peptide strongly interacts with the LPS of Gram-negative bacteria and folds upon interaction into a kinked helix.

Conclusion

Our results show that it is possible to widen the activity spectrum of an antimicrobial peptide by subtle changes of the primary structure. TB_KKG6A, having a simple composition, a broad spectrum of antimicrobial activity and a very low hemolytic activity, is a promising candidate for the design of novel antimicrobial peptides.

General significance

The activity of antimicrobial peptides is strongly related to the ability of the peptide to interact and break the bacterial membrane. Our studies on TB_KKG6A indicate that efficient interactions with LPS can be achieved when the peptide is not perfectly amphipathic, since this feature seems to help the toroidal pore formation process.     

Characteristics of Novel Insect Defensin-Based Membrane-Disrupting Trypanocidal Peptides

Synthetic D- and L-amino acid type cationic 9-mer peptides (all sequences were synthesized as D- or L-amino acids) derived from the active sites of insect defensins were tested for their ability to modify the growth of blood-stream form African trypanosomes in vitro. One of them, the D-type peptide A (RLYLRIGRR-NH₂), irreversibly suppressed proliferation of the Trypanosoma brucei brucei GUTat3.1 parasite. The presence of negatively charged phosphatidylserine on the surface of the parasites was demonstrated, suggesting electrostatic interaction between the peptide and the phospholipids. Furthermore, this peptide was found to alter trypanosome membrane-potentials significantly, an effect apparently due to the removal of the parasite’s plasma membrane. The potential toxic effects of D-peptide A on mammalian cells was assessed using human brain microvascular endothelial cells. Only minor effects were found when the endothelial cells were exposed for 16 h to peptide concentrations of less than 200 μM. These findings suggest that insect defensin-based peptides represent a potentially new class of membrane-disrupting trypanocidal drugs.     

Novel systemically active galanin receptor 2 ligands in depression‐like behavior

Neuropeptide galanin and its three G‐protein coupled receptors, galanin receptor type 1–galanin receptor type 3 (GalR1–GalR3), are involved in the regulation of numerous physiological and disease processes, and thus represent tremendous potential in neuroscience research and novel drug lead development. One of the areas where galanin is involved is depression. Previous studies have suggested that activation of GalR2 leads to attenuation of depression‐like behavior. Unfortunately, lack of in vivo usable subtype specific ligands hinders testing the role of galanin in depression mechanisms. In this article, we utilize an approach of increasing in vivo usability of peptide‐based ligands, acting upon CNS. Thus, we have synthesized a series of novel systemically active galanin analogs, with modest preferential binding toward GalR2. We have shown that specific chemical modifications to the galanin backbone increase brain levels upon i.v. injection of the peptides. Several of the new peptides, similar to a common clinically used antidepressant medication imipramine, exerted antidepressant‐like effect in forced swim test, a mouse model of depression, at a surprisingly low dose range (< 0.5 mg/kg). We chose one of the peptides, J18, for more thorough study, and showed its efficacy also in another mouse depression model (tail suspension test), and demonstrated that its antidepressant‐like effect upon i.v. administration can be blocked by i.c.v. galanin receptor antagonist M35. The effect of the J18 was also abolished in GalR2KO animals. All this suggests that systemically administered peptide analog J18 exerts its biological effect through activation of GalR2 in the brain. The novel galanin analogs represent potential drug leads and a novel pharmaceutical intervention for depression.

     

Rational design of highly stabilized and selective adrenomedullin analogs

The peptide hormone adrenomedullin (ADM) consists of 52 amino acids with a disulfide bond and an amidated C-terminus. Due to the vasodilatory and cardioprotective effects, the agonistic activity of the peptide on the adrenomedullin 1 receptor (AM₁R) is of high pharmacological interest. However, the wild-type peptide shows low metabolic stability leading to rapid degradation in the cardiovascular system. Previous work by our group has identified proteolytic cleavage sites and demonstrated stabilization of ADM by lipidation, cyclization, and N-methylation. Nevertheless, these ADM analogs showed reduced activity and subtype selectivity toward the closely related calcitonin gene-related peptide receptor (CGRPR). Here, we report on the rational development of ADM derivatives with increased proteolytic stability and high receptor selectivity. Stabilizing motifs, including lactamization and lipidation, were evaluated regarding AM₁R and CGRPR activation. Furthermore, the central DKDK motif of the peptide was replaced by oligoethylene glycol linkers. The modified peptides were synthesized by Fmoc/t-Bu solid-phase peptide synthesis and receptor activation of AM₁R and CGRPR was measured by cAMP reporter gene assay. Peptide stability was tested in human blood plasma and porcine liver homogenate and analyzed by RP-HPLC and MALDI-ToF mass spectrometry. Combination of the favorable lactam, lipidation, ethylene glycol linker, and previously described disulfide mimetic resulted in highly stabilized analogs with a plasma half-life of more than 144 h. The compounds display excellent AM₁R activity and wild-type-like selectivity toward CGRPR. Additionally, dose-dependent vasodilatory effects of the ADM derivatives lasted for several hours in rodents. Thus, we successfully developed an ADM analog with long-term in vivo activity.     

Purification and identification of a novel peptide derived from by-products fermentation of spiny head croaker (Collichthys lucidus) with antifungal effects on phytopathogens

In our previous study, spiny head croaker (Collichthys lucidus) by-products fermentation (SBF) products demonstrated plant growth promoting effects. In this study, the antimicrobial activities of SBF against plant pathogenic fungi and food-borne bacteria were evaluated. An active antifungal fraction SBF-3-1 was isolated from SBF using size exclusion chromatography and high performance liquid chromatography. Seven short peptides, i.e., LDEGW, DSFDFK, LDGW, FPDL, YNLDFK, FDGF and LDFE, plus one 22 residues peptide TFNTPAMYVAIQAVLSLYASGR were identified in SBF-3-1 by liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI-Q-TOF-MS/MS). The peptide TFNTPAMYVAIQAVLSLYASGR (named as SBF-3-1p), a potential antimicrobial peptide predicted by the antimicrobial peptide database (APD, http://aps.unmc.edu/AP/main.php), was further characterized. The prediction showed that the generation of SBF-3-1p could be associated with the action of trypsin and glutamyl endopeptidase, two proteases with specific cleavage sites on the carboxyl side of arginine (R) and lysine (K), and on the carboxyl side of Glu (E), respectively. According to the structural modeling, SBF-3-1p contained one α-helix and two loop regions. The synthesized SBF-3-1p exhibited strong activity against plant pathogenic fungus Pestalotiopsis sp. Therefore, SBF should be a good source of antifungal peptide, and could be used as natural fungicides in agriculture.     

Synthesis of shorter active analogues of Bactenecin7: The effect of change of N-terminal configuration on antimicrobial activity

Bactenecin7, a cationic antibacterial peptide, contains a repeating region of Xaa-Pro-Arg-Pro (Xaa = hydrophobic residues). A series of peptides, Xaa-Pro-Arg-Pro (Xaa = D-Ala, D-Leu, D-Val, D-Phe and D-Lys) were synthesized to investigate the effect of change of N-terminal configuration on antimicrobial activity. The conformationalpreferences of these peptides in water and TFE were examined by circular dichroism. All the synthetic peptides with D-amino acid substitution at N-terminal showed potent antifungal activity against Aspergillus niger, Aspergillus flavus and Fusarium moniliforme at the concentration level of 8–10 g ml^-1. But the same tetrapeptides were unable to kill or suppress the growth of gram-negativeand gram-positive bacteria such as Escherichia coli HB101, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus even at the concentration level of 400 g ml^-1. The present study reveals that the change of configuration at the N-terminal of tetrapeptide has negative impact on antibacterial activity but enhanced antifungal activity.     

Calorimetric and spectroscopic examination of the solution phase structures of prekallikrein binding domain peptides of high molecular weight kininogen

Unique sequence-binding sites are exposed on the surface of high molecular weight kininogen which complex prekallikrein or factor XI with high affinity and specificity. A sequence comprising 31 residues of the mature kininogen molecule (Asp565-Lys595) retains full binding activity for prekallikrein (K _D =20 nM) and assumes a complex folded structure in solution which is stabilized by long-range interactions between N- and C-terminal residues. The sequence Trp569-Lys595 (27 residues) shows only 28% of this binding affinity and lacks the key structural features required for protein recognition (Scarsale, J. N., and Harris, R. B.,J. Prot. Chem. 9, 647–659, 1990). We were thus able to predict that N- or C-terminal truncations of the binding-site sequence would disrupt the conformational integrity required for binding. Two new peptides of 20- and 22- residues have now been synthesized and their solution phase structures examined. These peptides are N- and C-terminal truncations, respectively, of the 27-residue sequence and correspond to the sequences Asp576-Lys595 and Trp569-Asp590 of high molecular weight kininogen. The results of fluorescence emission and circular dichroism (CD) spectroscopies in the range 25–90°C and from differential scanning calorimetry (DSC) all substantiate the idea that the C-terminal truncation peptide binds prekallikrein 35-fold poorer than the 31-residue peptide because it is relatively unoredered and possesses a less stable structure. Surprisingly, the N-terminal truncation peptide (20-mer) shows structural stability even at elevated temperatures and, like the 31-residue peptide, undergoes cold-induced denaturation observable in the DSC. 2D-NMR analysis of the 20-residue peptide revealed two distinct structures; one conformer possesses a more compact, folded structure than the other. However, the predicted structures assumed by either conformer are very different from those of either the 31- or 27-residue peptides. Hence, the binding affinity of the 20-residue peptide is 60-fold poorer than that for the 31-residue peptide because it assumes a nonproductive binding conformation(s).