Protein and Peptide Letters - Volume 20, Issue 6, 2013

Tachykinins (TKs) and their structurally related peptides constitute the largest peptide superfamily in the animal kingdom. TKs have been shown to play various physiological roles not only as major brain-gut peptides but also as endocrine-paracrine hormones in chordates and exocrine factors in amphibians. Recent studies have also revealed that the biological roles of TKs as brain-gut peptides and endocrine-paracrine factors are essentially conserved in protochordates, and that alternative splicing mechanism in mammalian TK genes were established during the evolution of vertebrates. Protostomes possess two structurally and functionally different peptides; invertebrate TKs (inv-TKs) serve as toxin-like compounds secreted from the salivary gland of several organisms, whereas TK-related peptides (TKRPs) are functional counterparts for chordate TKs. Additionally, a TKRP-like sequence was detected in a diploblastic organism. The dramatic difference in structural organizations between TKRP precursors and chordate TK precursors clearly indicates the distinct evolutionary processes of TKs and TKRPs. Despite high sequence homology, TK receptors manifest selective affinity to their endogenous ligands, while TKRPs exhibit redundant activity at their receptors. Moreover, in addition to nociceptive, inflammatory, and contractile effects as brain-gut peptides, a number of studies have revealed novel biological effects of TKs on the hypothalamus and genital organs, revealing the biological roles of TKs as pivotal regulators of reproduction. These findings shed light on complicated evolutionary lineages of both structures and functions of the TK-TKRP superfamily and their receptors. In this review, we present basic and latest knowledge of the TK-TKRP superfamily with various points of view.

The purpose of this review is to highlight the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in a range of physiological and behavioral processes of gastropod molluscs, Helix and Lymnaea. Since its discovery in 1989 PACAP has become increasingly recognized for its important and diversified roles in the central and peripheral nervous system and in several peripheral organs of a variety of vertebrate and invertebrate species. Twenty-two years after its discovery, PACAP is now one of the most extensively studied of the neuropeptides. This review surveys the importance of PACAP and PACAP-like peptides in invertebrates, focusing mainly on the gastropod molluscs. The relevance of studies on lower vertebrates and invertebrates, which do not have a pituitary gland, is to contribute to the unraveling of fundamental effects of PACAP or PACAP-like peptides and to provide a comparative view.

C. elegans has 302 neurons (in the adult hermaphrodite) and this simple nervous system harbours over 250 neuropeptides. Neuropeptides are a class of signalling molecule implicated in key physiological roles and thus confer a surprising level of complexity to signalling in this nematode. Indeed, it is probable that most, if not all, sensory, motor and interneurons, in C. elegans synthesise and release at least one neuropeptide but that many neurons synthesise an array of neuropeptides. In this review neuropeptides and their receptors with specific roles in feeding, metabolism, reproduction and locomotion are discussed. It is noted that the majority of C. elegans neuropeptides do not yet have defined roles and their cognate receptors have not yet been identified. Future studies will serve to provide further fundamental insight into how neuropeptide signalling can underpin animal behaviour.

In 1977 the neuropeptide FMRFamide was isolated from the clam, Macrocallista nimbosa. Since then several hundred FMRFamide-related peptides (FaRPs) have been isolated from invertebrate animals. Precursors to the FaRPs likely arose in the cnidarians. With the transition to a bilateral body plan FaRPs became a fixture in the invertebrate phyla. They have come to play a critical role as neurotransmitters, neuromodulators, and neurohormones. FaRPs regulate a variety of body functions including, feeding, digestion, circulation, reproduction, movement. The evolution of the molecular form and function of these omnipresent peptides will be considered.

Peptides are known to play important developmental and physiological roles in signaling. The rich diversity of peptides, with functions as diverse as intercellular communication, neurotransmission and signaling that spatially and temporally controls axis formation and cell differentiation, hints at the wealth of information passed between interacting cells. Little is known about peptides that control developmental processes such as cell differentiation and pattern formation in metazoans. The cnidarian Hydra is one of the most basic metazoans and is a key model system for study of the peptides involved in these processes. We developed a novel peptidomic approach for the isolation and identification of functional peptide signaling molecules from Hydra (the Hydra Peptide Project). Over the course of this project, a wide variety of novel neuropeptides were identified. Most of these peptides act directly on muscle cells and their functions include induction of contraction and relaxation. Some peptides are involved in cell differentiation and morphogenesis. Moreover, epitheliopeptides that are produced by epithelial cells were originally identified in Hydra. Some of these epitheliopeptides exhibit morphogen-like activities, whereas others are involved in regulating neuron differentiation, possibly through neuron-epithelial cell interactions. We also describe below our high-throughput reverse-phase nano-flow LCMALDI- TOF-MS-MS approach, which has proved a powerful tool for the discovery of novel peptide signaling molecules in Hydra.

A number of unique challenges are inherent to the study of neuropeptides (NPs), both in determining their molecular structure and their function. Traditional studies follow a model in which novel NPs are discovered and identified, then investigated for function. These studies frequently use biochemical techniques that can be imprecise and cumbersome. Mass spectrometry (MS)-based tools are becoming important not only in precisely determining the identity of a NP or quantifying a compound with a known sequence, but also in studies where identity and putative function can be determined simultaneously. Tools based on MS and tandem MS (MS-MS) have been developed, both with isotope labeling strategies and label-free methods, that allow accurate quantitation of NP changes associated with behavior or physiological manipulation, concurrent with identification of sequence. MS and MS-MS have also been implemented with sampling methods that incorporate temporal or spatial information while determining functional role of a NP, such as microdialysis (MD) and imaging mass spectrometry (IMS). These advances in MS and sampling techniques allow investigation of a particular biological phenomenon to guide studies aimed to identify and characterize NPs. Permitting function to drive identification of relevant compounds allows for a broader understanding of the molecular underpinnings of these events. The NPs thus identified can then be validated with more conventional techniques, and successive iterations of identification and function determination will provide rich information about these compounds. This function-driven discovery of NPs using MS-based techniques is an important new approach for their study.

Attachment to host cell surfaces is a crucial step in bacterial infections. This step is mediated by important virulence factors termed adhesins which are protein in nature. Non-covalent interactions play an important role in the structural stability of protein molecules. In the present study, the roles played by cation-π interactions in the adhesion proteins of Gram negative bacilli, Gram negative cocci and Gram positive cocci are systematically analyzed. There are significant differences in the pattern of interactions and environmental preferences like secondary structure, solvent accessibility, and stabilization centers for the amino acid residues which are involved in interactions. Among the cationic residues the role of Arg is significant in Gram negative group, while in the case of Gram positive cocci the contribution from Lys is found to be important. These results might be useful for understanding the stability patterns of adhesins in different groups of pathogenic bacteria.

In the circulatory system, serum albumin (SA) is an important transporter of the majority of molecules with biological activity. We focused the current study on the anti-inflammatory compound, o-alkylselenenylated benzoic acid (ALKSEBEA), to determine its ability to access SA. Herein, we employed experimental procedures (fluorescence studies, Raman spectroscopy) and docking study on SA obtained from the Protein Data Bank and key conformers obtained from molecular dynamics simulations. The results show that ALKSEBEA accesses SA using a cooperative behavior according to fluorescence studies. In addition, the Raman results indicate that the ligand binding affects the backbone constituents. These results were confirmed by docking simulations tested on several SA conformers, which showed that ALKSEBEA bound on several sites on SA via π-π or π-cation interactions and that the ligand reaches other binding sites, where aromatic and basic residues as well as the backbone residues are involved.

Porcine β defensin 2(pBD2)is a cationic 37-amino acid antimicrobial peptide with disulfide bonds. Synthetic pBD2 had broad antimicrobial activity against pathogenic bacteria, and thus pBD2 could be a good candidate as a bactericidal agent for pigs. This study reported the successful recombinant expression of pBD2 in Escherichia coli and analysis of its antimicrobial activity, its hemolytic activity, salt-tolerance and thermal stability as well. The pBD2 gene, obtained by RT-PCR using the tongue total RNA as a template and cloned into pET30a expression vector, was transformed into E. coli BL21 (DE3) plysS. The recombinant pBD2 was expressed after induction by IPTG and purified by His tag affinity column with 95% purity. The recombinant pBD2 exhibited antimicrobial activity against both Gram-positive S. aureus and Gram-negative E. coli including the multi-resistant E. coli. The minimum inhibitory concentration (MIC) of recombinant pBD2 against tested bacteria was 10 μg-mL, and the recombinant pBD2 could kill 50% E. coli at 14.39 μg-mL and S. aureus at 21.1 μg-mL. In addition, pBD2 showed low hemolytic activity, salt-tolerance and thermal stability, the properties would be important for its application in practice.

This work deals with the use of predictors to identify useful B-cell linear epitopes to develop immunoassays. Experimental techniques to meet this goal are quite expensive and time consuming. Therefore, we tested 5 free, online prediction methods (AAPPred, ABCpred, BcePred, BepiPred and Antigenic) widely used for predicting linear epitopes, using the primary structure of the protein as the only input. We chose a set of 65 experimentally well documented epitopes obtained by the most reliable experimental techniques as our true positive set. To compare the quality of the predictor methods we used their positive predictive value (PPV), i.e. the proportion of the predicted epitopes that are true, experimentally confirmed epitopes, in relation to all the epitopes predicted. We conclude that AAPPred and ABCpred yield the best results as compared with the other programs and with a random prediction procedure. Our results also indicate that considering the consensual epitopes predicted by several programs does not improve the PPV.