The iron-binding protein lactoferrin is a multifunctional protein that has antibacterial, antifungal, antiviral, antitumour, anti-inflammatory, and immunoregulatory properties. All of these additional properties appear to be related to its highly basic N-terminal region. This part of the protein can be released in the stomach by pepsin cleavage at acid pH. The 25-residue antimicrobial peptide that is released is called lactoferricin. In this work, we review our knowledge about the structure of the peptide and attempt to relate this to its many functions. Microcalorimetry and fluorescence spectroscopy data regarding the interaction of the peptide with model membranes show that binding to net negatively charged bacterial and cancer cell membranes is preferred over neutral eukaryotic membranes. Binding of the peptide destabilizes the regular membrane bilayer structure. Residues that are of particular importance for the activity of lactoferricin are tryptophan and arginine. These two amino acids are also prevalent in "penetratins", which are regions of proteins or synthetic peptides that can spontaneously cross membranes and in short hexapeptide antimicrobial peptides derived through combinatorial chemistry. While the antimicrobial, antifungal, antitumour, and antiviral properties of lactoferricin can be related to the Trp/Arg-rich portion of the peptide, we suggest that the anti-inflammatory and immunomodulating properties are more related to a positively charged region of the molecule, which, like the alpha- and beta-defensins, may act as a chemokine. Few small peptides are involved in as wide a range of host defense functions as bovine and human lactoferricin.

The interaction of several tryptophan (Trp)-rich cationic antimicrobial peptides with membranes was investigated. These peptides included tritrpticin, indolicidin, lactoferricin B (Lfcin B), and a shorter fragment of lactoferricin (LfcinB4-9). The average environment of the Trp residues of these peptides was assessed from their fluorescence properties, both the wavelength of maximal emission as well as the red edge effect. The insertion of the peptides into vesicles of differing composition was examined using quenching of the Trp fluorescence, with both soluble acrylamide and nitroxide-labelled phospholipids as well as by chemical modification of the Trp residues with N-bromosuccinimide. The results were consistent with the Trp side chains positioned mostly near the membrane-water interface. The extent of burial of the Trp side chains appears to be greater in vesicles containing phospholipids with the anionic phosphatidylglycerol headgroup. Leakage of the aqueous contents of liposomes was also measured using the 8-aminonaphthalene-1,3,6-trisulfonic acid--p-xylene-bis-pyridinium bromide assay. Tritrpticin, which demonstrated the greatest red edge shift, also displayed the largest amount of leakage from liposomes. Taken together, the results illustrate that cationic Trp-rich antimicrobial peptides preferentially disrupt large unilamellar vesicles with a net negative charge following their insertion into the interfacial region of the phospholipid bilayer.

Host defense antimicrobial peptides are key components of human innate immunity that plays an indispensible role in human health. While there are multiple copies of cathelicidin genes in horses, cattle, pigs, and sheep, only one cathelicidin gene is found in humans. Interestingly, this single cathelicidin gene can be processed into different forms of antimicrobial peptides. LL-37, the most commonly studied form, is not only antimicrobial but also possesses other functional roles such as chemotaxis, apoptosis, wound healing, immune modulation, and cancer metastasis. This article reviews recent advances made in structural and biophysical studies of human LL-37 and its fragments, which serve as a basis to understand their antibacterial, anti-biofilm and antiviral activities. High-quality structures were made possible by using improved 2D NMR methods for peptide fragments and 3D NMR spectroscopy for intact LL-37. The two hydrophobic domains in the long amphipathic helix (residues 2-31) of LL-37 separated by a hydrophilic residue serine 9 explain its cooperative binding to bacterial lipopolysaccharides (LPS). Both aromatic rings (F5, F6, F17, and F27) and interfacial basic amino acids of LL-37 directly interact with anionic phosphatidylglycerols (PG). Although the peptide sequences reported in the literature vary slightly, there is a consensus that the central helix of LL-37 is essential for disrupting superbugs (e.g., MRSA), bacterial biofilms, and viruses such as human immunodeficiency virus 1 (HIV-1) and respiratory syncytial virus (RSV). In the central helix, the central arginine R23 is of particular importance in binding to bacterial membranes or DNA. Mapping the functional roles of the cationic amino acids of the major antimicrobial region of LL-37 provides a basis for designing antimicrobial peptides with desired properties.

BackgroundWe recently provided evidence that HIV-1 enters HeLa-derived TZM-bl and lymphoid CEMss cells by fusing with endosomes, whereas its fusion with the plasma membrane does not proceed beyond the lipid mixing step. The mechanism of restriction of HIV-1 fusion at the cell surface and/or the factors that aid the virus entry from endosomes remain unclear.ResultsWe examined HIV-1 fusion with a panel of target cells lines and with primary CD4+ T cells. Kinetic measurements of fusion combined with time-resolved imaging of single viruses further reinforced the notion that HIV-1 enters the cells via endocytosis and fusion with endosomes. Furthermore, we attempted to deliberately redirect virus fusion to the plasma membrane, using two experimental strategies. First, the fusion reaction was synchronized by pre-incubating the viruses with cells at reduced temperature to allow CD4 and coreceptors engagement, but not the virus uptake or fusion. Subsequent shift to a physiological temperature triggered accelerated virus uptake followed by entry from endosomes, but did not permit fusion at the cell surface. Second, blocking HIV-1 endocytosis by a small-molecule dynamin inhibitor, dynasore, resulted in transfer of viral lipids to the plasma membrane without any detectable release of the viral content into the cytosol. We also found that a higher concentration of dynasore is required to block the HIV-endosome fusion compared to virus internalization.ConclusionsOur results further support the notion that HIV-1 enters disparate cell types through fusion with endosomes. The block of HIV-1 fusion with the plasma membrane at a post-lipid mixing stage shows that this membrane is not conducive to fusion pore formation and/or enlargement. The ability of dynasore to interfere with the virus-endosome fusion suggests that dynamin could be involved in two distinct steps of HIV-1 entry - endocytosis and fusion within intracellular compartments.

The apoptotic protein tBid is effective in promoting both leakage and lipid mixing in liposomes composed of cardiolipin and phosphatidylcholine at a molar ratio of 1:2 in the presence of calcium. When half of the phosphatidylcholine component of these liposomes is replaced with phosphatidylethanolamine, a lipid that promotes negative membrane curvature, the rates of both leakage and lipid mixing caused by tBid are substantially increased. Replacement of cardiolipin with phosphatidylglycerol, a lipid that is structurally similar to cardiolipin but does not promote negative membrane curvature in the presence of calcium, prevents the tBid from promoting leakage. The promotion of leakage by tBid is also inhibited by several substances that promote positive membrane curvature, including lysophosphatidylcholine, tritrpticin, a potent antimicrobial peptide, and cyclosporin A, a known inhibitor of cytochrome c release from mitochondria. We directly measured the effect of tBid on membrane curvature by 31 P NMR. We found that tBid promotes the formation of highly curved non-lamellar phases. All of these data are consistent with the hypothesis that tBid promotes negative curvature, and as a result it destabilizes bilayer membranes. Bcl-X L inhibits leakage and lipid mixing induced by tBid. Bcl-X L is anti-apoptotic. It reduces the promotion of non-bilayer phases by tBid, although by itself Bcl-X L is capable of promoting their formation. Bcl-X L has little effect on liposomal integrity. Our results suggest that the anti-apoptotic activity of Bcl-X L is not a consequence of its interaction with membranes, but rather with other proteins, such as tBid.

We have previously shown that two synthetic antimicrobial peptides with alternating alpha- and beta-amino acid residues, designated simply as alpha/beta-peptide I and alpha/beta-peptide II, had toxicity toward bacteria and affected the morphology of bacterial membranes in a manner that correlated with their effects on liposomes with lipid composition similar to those of the bacteria. In the present study we account for the weak effects of alpha/beta-peptide I on liposomes or bacteria whose membranes are enriched in phosphatidylethanolamine (PE) and why such membranes are particularly susceptible to damage by alpha/beta-peptide II. The alpha/beta-peptide II has marked effects on unilamellar vesicles enriched in PE causing vesicle aggregation and loss of their internal aqueous contents. The molecular basis of these effects is the ability of alpha/beta-peptide II to induce phase segregation of anionic and zwitterionic lipids as shown by fluorescence and differential scanning calorimetry. This phase separation could result in the formation of defects through which polar materials could pass across the membrane as well as form a PE-rich membrane domain that would not be a stable bilayer. alpha/beta-Peptide II is more effective in this regard because, unlike alpha/beta-peptide I, it has a string of two or three adjacent cationic residues that can interact with anionic lipids. Although alpha/beta-peptide I can destroy membrane barriers by converting lamellar to non-lamellar structures, it does so only weakly with unilamellar vesicles or with bacteria because it is not as efficient in the aggregation of these membranes leading to the bilayer-bilayer contacts required for this phase conversion. This study provides further understanding of why alpha/beta-peptide II is more toxic to micro-organisms with a high PE content in their membrane as well as for the lack of toxicity of alpha/beta-peptide I with these cells, emphasizing the potential importance of the lipid composition of the cell surface in determining selective toxicity of anti-microbial agents.

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 alpha-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 3(10)/alpha helix, whereas the shortest peptides tend to adopt a beta-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.

Antiviral drugs targeting viral proteins often result in prompt selection for resistance. Moreover, the number of viral targets is limited. Novel antiviral targets are therefore needed. The unique characteristics of fusion between virion envelopes and cell membranes may provide such targets. Like all fusing bilayers, viral envelopes locally adopt hourglass-shaped stalks during the initial stages of fusion, a process that requires local negative membrane curvature. Unlike cellular vesicles, however, viral envelopes do not redistribute lipids between leaflets, can only use the energy released by virion proteins, and fuse to the extracellular leaflets of cell membranes. Enrichment in phospholipids with hydrophilic heads larger than their hydrophobic tails in the convex outer leaflet of vesicles favors positive curvature, therefore increasing the activation energy barrier for fusion. Such phospholipids can increase the activation barrier beyond the energy provided by virion proteins, thereby inhibiting viral fusion. However, phospholipids are not pharmacologically useful. We show here that a family of synthetic rigid amphiphiles of shape similar to such phospholipids, RAFIs (rigid amphipathic fusion inhibitors), inhibit the infectivity of several otherwise unrelated enveloped viruses, including hepatitis C and HSV-1 and -2 (lowest apparent IC 50 48 nM), with no cytotoxic or cytostatic effects (selectivity index > 3,000) by inhibiting the increased negative curvature required for the initial stages of fusion.lipid bilayer fusion | DNA virus | RNA virus | Sindbis virus | nucleosides