Antimicrobial peptides: natural templates for synthetic membrane-active compounds

The innate immunity of multicellular organisms relies in large part on the action of antimicrobial peptides (AMPs) to resist microbial invasion. Crafted by evolution into an extremely diversified array of sequences and folds, AMPs do share a common amphiphilic 3-D arrangement. This feature is directly linked with a common mechanism of action that predominantly (although not exclusively) develops upon interaction of peptides with cell membranes of target cells. This minireview reports on current understanding of the modes of interaction of AMPs with biological and model membranes, especially focusing on recent insights into the folding and oligomerization requirements of peptides to bind and insert into lipid membranes and exert their antibiotic effects. Given the potential of AMPs to be developed into a new class of anti-infective agents, emphasis is placed on how the information on peptide-membrane interactions could direct the design and selection of improved biomimetic synthetic peptides with antibiotic properties.     

Antimicrobial skin peptides and proteins

Human skin is permanently exposed to microorganisms, but rarely infected. One reason for this natural resistance might be the existence of a ‘chemical barrier’ consisting in constitutively and inducibly produced antimicrobial peptides and proteins (AMPs). Many of these AMPs can be induced in vitro by proinflammatory cytokines or bacteria. Apart from being expressed in vivo in inflammatory lesions, some AMPs are also focally expressed in skin in the absence of inflammation. This suggests that non-inflammatory stimuli of endogenous and/or exogenous origin can also stimulate AMP synthesis without inflammation. Such mediators might be ideal ‘immune stimulants’ to induce only the innate antimicrobial skin effector molecules without causing inflammation. Received 9 August 2005; received after revision 21 October 2005; accepted 16 November 2005     

The Sushi peptides: structural characterization and mode of action against Gram-negative bacteria

The compositional difference in microbial and human cell membranes allows antimicrobial peptides to preferentially bind microbes. Peptides which specifically target lipopolysaccharide (LPS) and palmitoyl-oleoyl-phosphatidylglycerol (POPG) are efficient antibiotics. From the core LPS-binding region of Factor C, two 34-mer Sushi peptides, S1 and S3, were derived. S1 functions as a monomer, while S3 is active as a dimer. Both S1 and S3 display detergent-like properties in disrupting LPS aggregates, with specificity for POPG resulting from electrostatic and hydrophobic forces between the peptides and the bacterial lipids. During interaction with POPG, the S1 transitioned from a random coil to an α-helix, while S3 resumed a mixture of α-helix and β-sheet structures. The unsaturated nature of POPG confers fluidity and enhances insertion of the peptides into the lipid bilayer, causing maximal disruption of the bacterial membrane. These parameters should be considered in designing and developing new generations of peptide antibiotics with LPS-neutralizing capability. Received 2 October 2007; received after revision 2 November 2007; accepted 4 December 2007 J. L. Ding, B. Ho: Co-senior authors.     

Aspects of measurement and analysis of regulatory peptides

Summary Although almost all methods of mass measurement of regulatory peptides still depend on the high affinity antibody, the traditional Yalow and Berson radioimmunoassay technique is becoming outdated. Pure monoclonal antibodies allow excess antibody two site assay techniques with a variety of different labels (preferentially non-radioactive) of great sensitivity and speed. The large amounts of particular monoclonal antibodies available allow several different laboratories to use the same reagents and have increased comparability. Unfortunately many regulatory peptides exist in multiple molecular forms and attention must be paid to antibody region specificity. Improved methods of extraction of regulatory peptides from plasma tissue allow more accurate quantitation. New techniques for rapid high resolution chromatography make distinction of different molecular forms much easier than hitherto. Better education in techniques and/or attention to inter-assay standards are necessary to improve the comparability of regulatory peptide measurement in the future.     

Cell-penetrating peptides: tools for intracellular delivery of therapeutics

The main problem of therapeutic efficiency lies in the crossing of cellular membranes. Therefore, significant effort is being made to develop agents which can cross these barriers and deliver therapeutic agents into cellular compartments. In recent years, a large amount of data on the use of peptides as delivery agents has accumulated. Several groups have published the first positive results using peptides for the delivery of therapeutic agents in relevant animal models. These peptides, called cell-penetrating peptides (CPPs), are short peptides (fewer than 30 residues) with a net positive charge and acting in a receptor- and energy-independent manner. Here, we give an extensive review of peptide-mediated delivery systems and discuss their applications, with particular focus on the mechanisms leading to cellular internalization.Received 14 March 2005; received after revision 25 April 2005; accepted 28 April 2005     

Antimicrobial activity in the skin of the channel catfish Ictalurus punctatus: characterization of broad-spectrum histone-like antimicrobial proteins

Three antibacterial proteins were isolated from acid extracts of channel catfish (Ictalurus punctatus) skin by cation exchange chromatography and reverse-phase high-pressure liquid chromatography. The molecular masses of the proteins were 15.5, 15.5 and 30 kD as determined by SDS-polyacrylamide gel electrophoresis. Mass spectrometry, amino acid composition and amino acid sequence data suggest that the most abundant protein is closely related to histone H2B. The H2B-like protein was inhibitory to Aeromonas hydrophila and Saprolegnia spp., which are important bacterial and fungal pathogens of fish. These findings suggest that histones may be important defensive molecules in fish. Received 22 December 1997; received after revision 5 March 1998; accepted 5 March 1998     

The effect of corticotropin-releasing factor and pro-opiomelanocortin-derived peptides on the phagocytosis of molluscan hemocytes

The effect of corticotropin-releasing factor (CRF) and pro-opiomelanocortin (POMC)-derived peptides on hemocyte phagocytosis in two molluscs,Planorbarius corneus andViviparus ater was studied. The peptides and related fragments examined are those which have been shown to influence hemocyte motility in the two species. The results obtained revealed that the effects on phagocytosis are not directly correlated with previous findings on cell motility. Furthermore, the mode of action of an individual peptide could be species-specific and dose-dependent. The relationships between peptides, locomotion and phagocytosis in these molluscs are discussed.     

Lipopeptaibols, a novel family of membrane active, antimicrobial peptides

Lipopeptaibols are members of a novel group of naturally occurring, short peptides with antimicrobial activity, characterized by a lipophilic acyl chain at the N-terminus, a high content of the turn/helix forming α-aminoisobutyric acid and a 1,2-amino alcohol at the C-terminus. The amino acid sequences range from 6 to 10 residues and the fatty acyl moieties from 8 to 15 carbon atoms. The peptide portion of lipopeptaibols can be shorter than those of the nonlipidated peptaibols that range from 10 to 19 amino acid residues. The longest peptides fold into a mixed 3₁₀/α helix, whereas the shortest peptides tend to adopt a β-turn/sheet structure. Using solution methodologies, a series of analogues of trichogin GA IV was synthesized which allowed determination of the minimal lipid chain and peptide main-chain lengths for the onset of membrane activity and exploitation of a number of spectroscopic techniques aimed at determining its preferred conformation under a variety of conditions and investigating in detail its mode of interaction with, and its effect on, the phospholipid membranes. Received 26 January 2001; received after revision 7 March 2001; accepted 15 March 2001     

Novel cytotoxic peptides from the tropical marine cyanobacteriumHormothamnion enteromorphoides 1. Discovery,isolation and initial chemical and biological characterization of the hormothamnins from wild and cultured material

A Caribbean cyanobacterium,Hormothamnion enteromorphoides, was found to produce a complex mixture of ichthyotoxic peptides, perhaps explaining the apparent absence of predation upon these potentially palatable life forms. Bioassay-guided fractionation was used to isolate these toxic and antimicrobial natural products, and a variety of techniques including HR FAB mass spectrometry, 2D-NMR, traditional hydrolysis-amino acid analysis, and several chemical reactions were used to define the basic structural features of the major peptide, hormothamnin A. Hormothamnin A is a cyclic undecapeptide containing six common and five uncommon or new amino acid residues. HPLC analyses indicate that the relative proportions of these peptide natural products remain relatively constant between different collection locations and years, however, they do vary seasonally. Clonal isolates of this cyanobacterium in culture produce the full spectrum of toxic peptides.     

Penaeidins, a family of antimicrobial peptides from penaeid shrimp (Crustacea, Decapoda)

The production of antimicrobial peptides represents a first-line host defense mechanism of innate immunity that is widespread in nature. Only recently such effectors were isolated in crustacean species, whereas numerous antimicrobial peptides have been characterized from other arthropods, both insects and chelicerates. This review presents findings on a family of antimicrobial peptides, named penaeidins, isolated from the shrimp Penaeus vannamei. Their structure and antimicrobial properties as well as their immune function will be discussed through analyses of penaeidin gene expression and peptide distribution upon microbial challenge. Received 21 January 2000; received after revision 10 March 2000; accepted 10 March 2000     

海洋活性肽研究的回顾与展望

海洋环境资源丰富,物种多样,是活性肽开发与研究的巨大宝库。本文分析了活性肽丰富的海洋来源及生物功用,并且对活性肽的自然来源途径和人工合成来源途径进行了论述,包括活性肽的分离、纯化、合成、筛选等方面,并强调了海洋活性肽在食品工业、医药学、畜牧业、水产养殖业、尤其是在环境保护中的应用现状与潜力,还从多角度、多层面对海洋活性肽的研究作出展望．     

Biosynthesis, immunity, regulation, mode of action and engineering of the model lantibiotic nisin

This review discusses the state-of-the-art in molecular research on the most prominent and widely applied lantibiotic, i.e., nisin. The developments in understanding its complex biosynthesis and mode of action are highlighted. Moreover, novel applications arising from engineering either nisin itself, or from the construction of totally novel dehydrated and/or lanthionine-containing peptides with desired bioactivities are described. Several challenges still exist in understanding the immunity system and the unique multiple reactions occurring on a single substrate molecule, carried out by the dehydratase NisB and the cyclization enzyme NisC. The recent elucidation of the 3-D structure of NisC forms the exciting beginning of further 3-D-structure determinations of the other biosynthetic enzymes, transporters and immunity proteins. Advances in achieving in vitro activities of lanthionine-forming enzymes will greatly enhance our understanding of the molecular characteristics of the biosynthesis process, opening up new avenues for developing unique and novel biocatalytic processes. Received 9 April 2007; received after revision 31 August 2007; accepted 28 September 2007     

Nervous control of smooth muscle by transmitters,cotransmitters and modulators

Conclusion The part played by peripheral neuroeffector control mechanisms has been underestimated. These are additional to central and ganglionic control mechanisms and are much more elaborate than originally thought. While the classical view is that the autonomic nervous system consists largely of antagonistic cholinergic and adrenergic nerves, about sixteen putative neurotransmitters have been proposed in autonomic nerves in the past few years, including various monoamines, polypeptides, purines and amino acids. Modulatory transmitter mechanisms have also been recognized, including prejunctional inhibition or enhancement of transmitter release, postjunctional modulation of transmitter action, and the secondary involvement of locally synthesized hormones and prostaglandins. The existence of more than one transmitter substance in some nerves is now widely recognized, and suggestions have been made about the ways that this can lead to differential peripheral control mechanisms at nerve terminals themselves. The cotransmitters always have synergistic actions on postjunctional effector cells, but two different operating mechanisms are postulated. 1) If both substances are stored in the same vesicles (for example, ACh or NA with ATP), release is closely parallel at all impulse frequencies. Upon release, the cotransmitter, in addition to having a direct action on postjunctional cells, may facilitate the action of the other transmitter and/or act as an inhibitor of its release. Differential actions at different impulse frequencies are achieved post-junctionally by ATP and NA acting via EJP-spike and spike-independent mechanisms, respectively. 2) If the two substances are stored in separate vesicle types (for example ACh or NA with some peptides), then differential release is possible at different impulse frequencies; the peptides released at higher frequencies modulate the role of the classical transmitter, by both prejunctional enhancement of its release and post-junctional facilitation of its action.     

A dynamic view of peptides and proteins in membranes

Biological membranes are highly dynamic supramolecular arrangements of lipids and proteins, which fulfill key cellular functions. Relatively few high-resolution membrane protein structures are known to date, although during recent years the structural databases have expanded at an accelerated pace. In some instances the structures of reaction intermediates provide a stroboscopic view on the conformational changes involved in protein function. Other biophysical approaches add dynamic aspects and allow one to investigate the interactions with the lipid bilayers. Membrane-active peptides fulfill many important functions in nature as they act as antimicrobials, channels, transporters or hormones, and their studies have much increased our understanding of polypeptide-membrane interactions. Interestingly several proteins have been identified that interact with the membrane as loose arrays of domains. Such conformations easily escape classical high-resolution structural analysis and the lessons learned from peptides may therefore be instructive for our understanding of the functioning of such membrane proteins. Received 11 March 2008; received after revision 2 May 2008; accepted 5 May 2008     

Co-localization of glucagon and pancreatic polypeptide in testudine pancreas

Summary Immunocytochemistry was carried out on sections of pancreas from the gopher tortoise,Gopherus polyphemus. Combined immunofluorescent and peroxidase-anti-peroxidase techniques showed unequivocally that some of the cells were immunoreactive for both glucagon and pancreatic polypeptide (PP). Antibodies directed against avian PP, bovine PP, and human PP all have a positive reaction. Co-localization of glucagon and PP in the pancreas of the gopher tortoise indicates that the occurrence of these hormones in the same cells is widespread in higher vertebrates.Acknowledgments. This work was supported in part by grants from the University of Cape Town, the M. Crossley bequest, the N. Atkinson bequest, and Herman and Caporn bequests. The generous gifts of antisera by Dr Chance and the late Dr Kimmel, and the gift of hormones by the Lilly Research Laboratories are gratefully acknowledged. The excellent technical assistance of J. Thompson and J. B. De Haan is also gratefully acknowledged. Please send all correspondence to the USA address.     

Developmental actions of natriuretic peptides in the brain and skeleton

The concept that atrial natriuretic peptide (ANP) and the closely related peptides BNP and CNP might be involved in the ontogeny of several organ systems emerged in the late 1980s. While many of the reported in vitro actions have not been examined in the context of organ development in vivo, recent studies demonstrate that mice which lack or overexpress natriuretic peptides or receptors exhibit pronounced skeletal growth defects. This article discusses how natriuretic peptides and other factors appear to regulate bone growth as an example of how natriuretic peptides might participate in the ontogeny of other organ systems. Evidence indicating that natriuretic peptides regulate neural development is then reviewed. Natriuretic peptides and receptors exhibit complex expression patterns in the developing nervous system, where they have been shown to act on neural cells as early as at the embryonic neural tube stage. Interestingly, both bone and brain growth appear to utilize primarily CNP and the CNP-specific type B receptor, and perhaps the type C receptor. In vitro data indicate that CNP may act on developing neurons, astrocytes and Schwann cells like a classical growth factor, regulating proliferation, patterning, phenotypic specification, survival and axonal pathfinding. Natriuretic peptides might also have roles in the vascularization of the embryonic brain, establishment of the blood-brain and blood-nerve barriers, and perhaps in nerve regeneration.Received 13 April 2004; received after revision 20 May 2004; accepted 27 May 2004     

Effects of enterally- and parenterally-administered bombesin on intestinal luminal tryptic activity and protein in the suckling rat

Summary Because of the presence of bombesin-like immunoreactivity in milk we investigated if enteral administration of bombesin affects the intestinal luminal content of trypsin and protein in 12-14-day-old rats. Bombesin (40 g/kg), given either orogastrically or subcutaneously, produced a significant elevation in the intestinal content of trypsin activity. Thus, enterally-administered bombesin can produce acute biologic effects in suckling rats.     

Participation of ACTH1–10 and ACTH4–10 on the melatonin modulation of benzodiazepine receptors in rat cerebral cortex

In this study, we examined the effect of intracerebroventricular (i.c.v) injection of melatonin and/or ACTH_1–10 and ACTH_4–10 on [³H]flunitrazepam binding sites in the cerebral cortex of hypophysectomized rats. Hypophysectomy increased the B_max (maximum number of binding sites) of benzodiazepine (BNZ) receptors for at least 7 days after surgery, without changing K_D (dissociation constant). The i.c.v. injection of melatonin to hypophysectomized rats significantly increased B_max, whereas the same doses of melatonin were ineffective in sham-operated animals. In both cases, K_D values were unchanged. The i.c.v injection of ACTH_1–10 to hypophysectomized animals significantly increased B_max, an effect that was enhanced by simultaneous i.c.v. injection of ACTH_1–10+melatonin, reaching higher values of B_max than the i.c.v. injection of these hormones individually. No significant changes in K_D values were found after ACTH_1–10 and/or melatonin administration. However, the i.c.v. injection of ACTH_4–10 to hypophysectomized rats did not change B_max, although it significantly increased K_D values, indicating a decrease in the BNZ binding affinity. Melatonin injection counteracted this effect of ACTH_4–10, returning K_D to the control value. Moreover, although the lower dose of i.c.v. melatonin used, 10 ng, was unable to modify B_max of BNZ binding in the ACTH_4–10-injected group, the higher dose, 20 ng, significantly increased B_max. The results suggest that these ACTH-derived peptides can modulate the effect of melatonin on brain benzodiazepine receptors.     

The effect of ethanol on the biosynthesis and regulation of opioid peptides

Summary Alcoholism and alcohol abuse are serious health problems. Alcohol is known to influence the activity of a number of biological systems, for example the hormonal and neuronal systems. One of the biological systems whose activity is greatly influenced by alcohol is the endogenous opiate system. Alcohol modifies the function of both opiate receptors and opioid peptides. In fact it has been proposed that many of the effects of ethanol are mediated by its effects on the endogenous opiate system. This review will present results from various laboratories on the effects of acute and chronic ethanol treatments on various species, and on the release, biosynthesis and post-translational processing of the endorphins, enkephalins and dynorphins, the three known families of endogenous opioid peptides. Furthermore, the effect of acute and chronic ethanol consumption on the -endorphin system in man, and the possible implications of the functional activity of the endogenous opiate system for the genetic predisposition to alcoholism will be discussed.