The Central PXXP Motif Is Crucial for PMAP-23 Translocation across the Lipid Bilayer.

PMAP-23, a cathelicidin-derived host defense peptide, does not cause severe membrane permeabilization, but exerts strong and broad-spectrum bactericidal activity. We have previously shown that it forms an amphipathic α-helical structure with a central hinge induced by the PXXP motif, which is implicated in the interaction of PMAP-23 with negatively charged bacterial membranes. Here, we studied the potential roles of the PXXP motif in PMAP-23 translocation across the lipid bilayer by replacing Pro residues with either α-helix former Ala (PMAP-PA) or α-helix breaker Gly (PMAP-PG). Although both PMAP-PA and PMAP-PG led to effective membrane depolarization and permeabilization, they showed less antimicrobial activity than wild-type PMAP-23. Interestingly, we observed that PMAP-23 crossed lipid bilayers much more efficiently than its Pro-substituted derivatives. The fact that the Gly-induced hinge was unable to replace the PXXP motif in PMAP-23 translocation suggests that the PXXP motif has unique structural properties other than the central hinge. Surface plasmon resonance sensorgrams showed that the running buffer almost entirely dissociated PMAP-23 from the membrane surface, while its Pro-substituted derivatives remained significantly bound to the membrane. In addition, kinetic analysis of the sensorgrams revealed that the central PXXP motif allows PMAP-23 to rapidly translocate at the interface between the hydrophilic and hydrophobic phases. Taken together, we propose that the structural and kinetic understanding of the PXXP motif in peptide translocation could greatly aid the development of novel antimicrobial peptides with intracellular targets by promoting peptide entry into bacterial cells.

Release pattern of dexamethasone after administration through an implant-mediated drug delivery device with an active plunger of super absorbent polymer.

The implant-mediated drug delivery system (IMDDS) is a novel, innovative device that allows drug delivery through bone marrow. The purpose of this study was to investigate the effect of an active plunger component made of super absorbent polymer (SAP) on the plasma concentration of dexamethasone released from the IMDDS. The IMDDSs were installed in a total of 18 rabbits. After complete healing, dexamethasone was loaded with the SAP active plunger and with water to cause expansion in the test group (n = 9), while only the drug was loaded in the control group, as per the original protocol (n = 9). The release patterns of each group were monitored for 2 weeks by measuring the plasma concentration of the drug. Both groups showed sustained release of drug. However, the test groups showed more rapid increase in plasma concentration and higher area under the curve (AUC) throughout the observation period. The incorporation of a SAP active plunger component in the IMDDS resulted in an increase in initial release of drug and higher bioavailability within the observation period of 2 weeks after dexamethasone administration.

Asymmetric Phosphatidylethanolamine Distribution Controls Fusion Pore Lifetime and Probability.

Little attention has been given to how the asymmetric lipid distribution of the plasma membrane might facilitate fusion pore formation during exocytosis. Phosphatidylethanolamine (PE), a cone-shaped phospholipid, is predominantly located in the inner leaflet of the plasma membrane and has been proposed to promote membrane deformation and stabilize fusion pores during exocytotic events. To explore this possibility, we modeled exocytosis using plasma membrane SNARE-containing planar-supported bilayers and purified neuroendocrine dense core vesicles (DCVs) as fusion partners, and we examined how different PE distributions between the two leaflets of the supported bilayers affected SNARE-mediated fusion. Using total internal reflection fluorescence microscopy, the fusion of single DCVs with the planar-supported bilayer was monitored by observing DCV-associated neuropeptide Y tagged with a fluorescent protein. The time-dependent line shape of the fluorescent signal enables detection of DCV docking, fusion-pore opening, and vesicle collapse into the planar membrane. Four different distributions of PE in the planar bilayer mimicking the plasma membrane were examined: exclusively in the leaflet facing the DCVs; exclusively in the opposite leaflet; equally distributed in both leaflets; and absent from both leaflets. With PE in the leaflet facing the DCVs, overall fusion was most efficient and the extended fusion pore lifetime (0.7 s) enabled notable detection of content release preceding vesicle collapse. All other PE distributions decreased fusion efficiency, altered pore lifetime, and reduced content release. With PE exclusively in the opposite leaflet, resolution of pore opening and content release was lost.

HIV virions sense plasma membrane heterogeneity for cell entry.

It has been proposed that cholesterol in host cell membranes plays a pivotal role for cell entry of HIV. However, it remains largely unknown why virions prefer cholesterol-rich heterogeneous membranes to uniformly fluid membranes for membrane fusion. Using giant plasma membrane vesicles containing cholesterol-rich ordered and cholesterol-poor fluid lipid domains, we demonstrate that the HIV receptor CD4 is substantially sequestered into ordered domains, whereas the co-receptor CCR5 localizes preferentially at ordered/disordered domain boundaries. We also show that HIV does not fuse from within ordered regions of the plasma membrane but rather at their boundaries. Ordered/disordered lipid domain coexistence is not required for HIV attachment but is a prerequisite for successful fusion. We propose that HIV virions sense and exploit membrane discontinuities to gain entry into cells. This study provides surprising answers to the long-standing question about the roles of cholesterol and ordered lipid domains in cell entry of HIV and perhaps other enveloped viruses.

Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein.

Fluorescence approaches have been widely used for elucidating the dynamics of protein-membrane interactions in cells and model systems. However, non-specific multi-site fluorescent labeling often results in a loss of native structure and function, and single cysteine labeling is not feasible when native cysteines are required to support a protein's folding or catalytic activity. Here, we develop a method using genetic incorporation of non-natural amino acids and bio-orthogonal chemistry to site-specifically label with a single fluorescent small molecule or protein the myristoyl-switch protein recoverin, which is involved in rhodopsin-mediated signaling in mammalian visual sensory neurons. We demonstrate reversible Ca(2+)-responsive translocation of labeled recoverin to membranes and show that recoverin favors membranes with negative curvature and high lipid fluidity in complex heterogeneous membranes, which confers spatio-temporal control over down-stream signaling events. The site-specific orthogonal labeling technique is promising for structural, dynamical, and functional studies of many lipid-anchored membrane protein switches.

The role of cholesterol in membrane fusion.

Cholesterol modulates the bilayer structure of biological membranes in multiple ways. It changes the fluidity, thickness, compressibility, water penetration and intrinsic curvature of lipid bilayers. In multi-component lipid mixtures, cholesterol induces phase separations, partitions selectively between different coexisting lipid phases, and causes integral membrane proteins to respond by changing conformation or redistribution in the membrane. But, which of these often overlapping properties are important for membrane fusion?-Here we review a range of recent experiments that elucidate the multiple roles that cholesterol plays in SNARE-mediated and viral envelope glycoprotein-mediated membrane fusion.

Line tension at lipid phase boundaries as driving force for HIV fusion peptide-mediated fusion.

Lipids and proteins are organized in cellular membranes in clusters, often called 'lipid rafts'. Although raft-constituent ordered lipid domains are thought to be energetically unfavourable for membrane fusion, rafts have long been implicated in many biological fusion processes. For the case of HIV gp41-mediated membrane fusion, this apparent contradiction can be resolved by recognizing that the interfaces between ordered and disordered lipid domains are the predominant sites of fusion. Here we show that line tension at lipid domain boundaries contributes significant energy to drive gp41-fusion peptide-mediated fusion. This energy, which depends on the hydrophobic mismatch between ordered and disordered lipid domains, may contribute tens of kBT to fusion, that is, it is comparable to the energy required to form a lipid stalk intermediate. Line-active compounds such as vitamin E lower line tension in inhomogeneous membranes, thereby inhibit membrane fusion, and thus may be useful natural viral entry inhibitors.

Supported lipid bilayers as models for studying membrane domains.

Supported lipid bilayers have been in use for over 30 years. They have been employed to study the structure, composition, and dynamics of lipid bilayer phases, the binding and distribution of soluble, integral, and lipidated proteins in membranes, membrane fusion, and interactions of membranes with elements of the cytoskeleton. This review focuses on the unique ability of supported lipid bilayers to study liquid-ordered and liquid-disordered domains in membranes. We highlight methods to produce asymmetric lipid bilayers with lipid compositions that mimic those of the extracellular and cytoplasmic leaflets of cell membranes and the functional reconstitution of membrane proteins into such systems. Questions related to interleaflet domain coupling and membrane protein activation have been addressed and answered using advanced reconstitution and imaging procedures in symmetric and asymmetric supported membranes with and without coexisting lipid phase domains. Previously controversial topics regarding anomalous and anisotropic diffusion in membranes have been resolved by using supported membrane approaches showing that the propensity of certain lipid compositions to form "rafts" are important but overlaid with "picket-fence" interactions that are imposed by a subtended cytoskeletal network.

HIV gp41-mediated membrane fusion occurs at edges of cholesterol-rich lipid domains.

Lipid rafts in plasma membranes have emerged as possible platforms for the entry of HIV and other viruses into cells. However, little is known about how lipid phase heterogeneity contributes to viral entry because of the fine-grained and still poorly understood complexity of biological membranes. We used model systems mimicking HIV envelopes and T cell membranes and found that raft-like liquid-ordered (Lo-phase) lipid domains were necessary and sufficient for efficient membrane targeting and fusion. Interestingly, membrane binding and fusion were low in homogeneous liquid-disordered (Ld-phase) and Lo-phase membranes, indicating that lipid phase heterogeneity is essential. The HIV fusion peptide preferentially targeted to Lo-Ld boundary regions and promoted full fusion at the interface between ordered and disordered lipids. Ld-phase vesicles proceeded only to hemifusion. Thus, we propose that edges but not areas of raft-like ordered lipid domains are vital for HIV entry and membrane fusion.

Cationic cell-penetrating peptide binds to planar lipid bilayers containing negatively charged lipids but does not induce conductive pores.

Using a cation-selective gramicidin A channel as a sensor of the membrane surface charge, we studied interactions of oligoarginine peptide R9C, a prototype cationic cell-penetrating peptide (CPP), with planar lipid membranes. We have found that R9C sorption to the membrane depends strongly on its lipid composition from virtually nonexistent for membranes made of uncharged lipids to very pronounced for membranes containing negatively charged lipids, with charge overcompensation at R9C concentrations exceeding 1 µM. The sorption was reversible as it was removed by addition of polyanionic dextran sulfate to the membrane bathing solution. No membrane poration activity of R9C (as would be manifested by increased bilayer conductance) was detected in the charged or neutral membranes, including those with asymmetric negative/neutral and negative/positive lipid leaflets. We conclude that interaction of R9C with planar lipid bilayers does not involve pore formation in all studied lipid combinations up to 20 µM peptide concentration. However, R9C induces leakage of negatively charged but not neutral liposomes in a process that involves lipid mixing between liposomes. Our findings suggest that direct traversing of CPPs through the uncharged outer leaflet of the plasma membrane bilayer is unlikely and that permeabilization necessarily involves both anionic lipids and CPP-dependent fusion between opposing membranes.

A three dimensional observation of palatal vault growth in children using mixed effect analysis: a 9 year longitudinal study.

The understanding of palatine vault growth in normal subjects is important to orthodontists. The aim of this study was to evaluate three dimensional (3D) longitudinal changes in the palatal vault from 6 to 14 years of age. Complete dental stone casts were biennially prepared for 50 subjects (25 girls and 25 boys) followed up from 6 to 14 years of age. Virtual casts were constructed using 3D laser scanning and reconstruction software. The reference gingival plane was constructed. The palatal heights were measured from a total of 12 quadrisectional points between the most gingival points of the palatal dentogingival junctions from the canine to the first molar. In addition, the palatal heights were measured from a total of 12 lateral and medial endpoints of the palatine rugae. The measurement changes over time were analyzed using a mixed-effect analysis. There were significant annual increases in all of the variables related to palatal height. However, the individual random variability at baseline was quite large. There was no significant sexual dimorphism in the linear measurements or in the annual increases as fixed effects in the model. During the observation period, increases in palatal vault height were significant in all regions. The growth pattern seemed to differ between genders even though it was not significant. More elaborate methodology is necessary to gain a better understanding of 3D palatal growth.

Mechanistic study of broadly neutralizing human monoclonal antibodies against dengue virus that target the fusion loop.

There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.

Cell-penetrating peptide induces leaky fusion of liposomes containing late endosome-specific anionic lipid.

Cationic cell-penetrating peptides (CPPs) are a promising vehicle for the delivery of macromolecular drugs. Although many studies have indicated that CPPs enter cells by endocytosis, the mechanisms by which they cross endosomal membranes remain elusive. On the basis of experiments with liposomes, we propose that CPP escape into the cytosol is based on leaky fusion (i.e., fusion associated with the permeabilization of membranes) of the bis(monoacylglycero)phosphate (BMP)-enriched membranes of late endosomes. In our experiments, prototypic CPP HIV-1 TAT peptide did not interact with liposomes mimicking the outer leaflet of the plasma membrane, but it did induce lipid mixing and membrane leakage as it translocated into liposomes mimicking the lipid composition of late endosome. Both membrane leakage and lipid mixing depended on the BMP content and were promoted at acidic pH, which is characteristic of late endosomes. Substitution of BMP with its structural isomer, phosphatidylglycerol (PG), significantly reduced both leakage of the aqueous probe from liposomes and lipid mixing between liposomes. Although affinity of binding to TAT was similar for BMP and PG, BMP exhibited a higher tendency to support the inverted hexagonal phase than PG. Finally, membrane leakage and peptide translocation were both inhibited by inhibitors of lipid mixing, further substantiating the hypothesis that cationic peptides cross BMP-enriched membranes by inducing leaky fusion between them.

Dengue virus ensures its fusion in late endosomes using compartment-specific lipids.

Many enveloped viruses invade cells via endocytosis and use different environmental factors as triggers for virus-endosome fusion that delivers viral genome into cytosol. Intriguingly, dengue virus (DEN), the most prevalent mosquito-borne virus that infects up to 100 million people each year, fuses only in late endosomes, while activation of DEN protein fusogen glycoprotein E is triggered already at pH characteristic for early endosomes. Are there any cofactors that time DEN fusion to virion entry into late endosomes? Here we show that DEN utilizes bis(monoacylglycero)phosphate, a lipid specific to late endosomes, as a co-factor for its endosomal acidification-dependent fusion machinery. Effective virus fusion to plasma- and intracellular- membranes, as well as to protein-free liposomes, requires the target membrane to contain anionic lipids such as bis(monoacylglycero)phosphate and phosphatidylserine. Anionic lipids act downstream of low-pH-dependent fusion stages and promote the advance from the earliest hemifusion intermediates to the fusion pore opening. To reach anionic lipid-enriched late endosomes, DEN travels through acidified early endosomes, but we found that low pH-dependent loss of fusogenic properties of DEN is relatively slow in the presence of anionic lipid-free target membranes. We propose that anionic lipid-dependence of DEN fusion machinery protects it against premature irreversible restructuring and inactivation and ensures viral fusion in late endosomes, where the virus encounters anionic lipids for the first time during entry. Currently there are neither vaccines nor effective therapies for DEN, and the essential role of the newly identified DEN-bis(monoacylglycero)phosphate interactions in viral genome escape from the endosome suggests a novel target for drug design.

Analysis of the solution structure of the human antibiotic peptide dermcidin and its interaction with phospholipid vesicles.

Dermcidin is a human antibiotic peptide that is secreted by the sweat glands and has no homology to other known antimicrobial peptides. As an initial step toward understanding dermcidin's mode of action at bacterial membranes, we used homonuclear and heteronuclear NMR to determine the conformation of the peptide in 50% trifluoroethanol solution. We found that dermcidin adopts a flexible amphipathic alpha-helical structure with a helix-hinge-helix motif, which is a common molecular fold among antimicrobial peptides. Spin-down assays of dermcidin and several related peptides revealed that the affinity with which dermcidin binds to bacterial-mimetic membranes is primarily dependent on its amphipathic alpha-helical structure and its length (>30 residues); its negative net charge and acidic pI have little effect on binding. These findings suggest that the mode of action of dermcidin is similar to that of other membrane-targeting antimicrobial peptides, though the details of its antimicrobial action remain to be determined.

Cell selectivity and anti-inflammatory activity of a Leu/Lys-rich alpha-helical model antimicrobial peptide and its diastereomeric peptides.

To investigate the effect of the number and distribution of d-amino acids introduced into non-cell-selective alpha-helical antimicrobial peptides on the cell selectivity, protease stability and anti-inflammatory activity, we synthesized an 18-meric Leu/Lys-rich alpha-helical model peptide (K(9)L(8)W) and d-amino acid-containing diastereomeric peptides. Increasing in cell selectivity of the peptides was increased in parallel with increasing in the number of d-amino acids introduced. Despite having the same number of d-amino acids, D(9)-K(9)L(8)W-1 had better cell selectivity than D(9)-K(9)L(8)W-2, indicating that a dispersed distribution of d-amino acids in diastereomeric peptides is more effective for cell selectivity than their segregated distribution. D(3)-K(9)L(8)W-2, D(6)-K(9)L(8)W, D(9)-K(9)L(8)W-1 and D(9)-K(9)L(8)W-2 showed complete resistance to tryptic digestion. Furthermore, K(9)L(8)W and all of its diastereomeric peptides significantly inhibited nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) mRNA expression and tumor necrosis factor-alpha (TNF-alpha) release in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells at a lower concentration than bactericidal concentration. The order of anti-inflammatory activity for the peptides was K(9)L(8)W approximately D(3)-K(9)L(8)W-1 approximately D(3)-K(9)L(8)W-2 approximately D(6)-K(9)L(8)W approximately D(9)-K(9)L(8)W-2>D(4)-K(9)L(8)W>D(9)-K(9)L(8)W-1. Increasing in hydrophobicity or alpha-helicity of the peptides was more closely correlated with increasing in hemolytic activity and anti-inflammatory activity than antimicrobial and LPS-disaggregation activities. Collectively, we successfully developed several d-amino acid-containing antimicrobial peptides (D(4)-K(9)L(8)W, D(6)-K(9)L(8)W and D(9)-K(9)L(8)W-1) with good cell selectivity, protease stability and potent anti-inflammatory activity. These antimicrobial peptides could serve as templates for the development of peptide antibiotics for the treatment of sepsis, as well as microbial infection.

Different modes of antibiotic action of homodimeric and monomeric bactenecin, a cathelicidin-derived antibacterial peptide.

The bactenecin is an antibacterial peptide with an intramolecular disulfide bond. We recently found that homodimeric bactenecin exhibits more potent antibacterial activity than the monomeric form and retains its activity at physiological conditions. Here we assess the difference in the modes of antibiotic action of homodimeric and monomeric bactenecins. Both monomeric and dimeric bactenecins almost completely killed both Staphylococcus aureus and E. coli within 10-30 min at concentrations of 8-16 muM. However, exposure to liposomes elicited an increase in the fluorescence quantum yield from a tryptophan-containing monomeric analog, while the homodimeric analog showed a significant reduction in fluorescence intensity. Moreover, unlike the monomer, the homodimer displayed apparent membrane-lytic activity enabling release of various sized dyes from liposomes, and rapidly and fully depolarized the S. aureus membrane. Together, our results suggest that homodimeric bactenecin forms pores in the bacterial membrane, while monomeric one penetrates through the membrane to target intracellular molecules/organelles. [BMB reports 2009; 42(9): 586-592].

Molecular interaction between kisspeptin decapeptide analogs and a lipid membrane.

Kisspeptin-10 is the C-terminal decapeptide amide of kisspeptin, an endogenous ligand for GPR54, and exhibits the same binding and agonist activity as the parent molecule. Although GPR54 is a membrane-embedded protein, details of the molecular interaction between kisspeptin-10 and lipid membranes remain unclear. Here, we performed a series of structural analyses using alanine-scanning analogs of kisspeptin-10 in membrane-mimetic medium. We found that there is a close correlation between lipid membrane binding and agonist activity. For instance, the F10A and non-amidated (NH2-->OH) analogs showed little or no GPR54-agonist activity and elicited no blue shift in tryptophan fluorescence. NMR analysis of kisspeptin-10 analog in DPC micelles revealed it to contain several tight turn structures, encompassing residues Trp3 to Phe10, but no helical conformation like that seen previously with SDS micelles. Together, our results suggest that kisspeptin-10 may activate GPR54 via a ligand transportation pathway incorporating a lipid membrane.

Effect of dimerization of a beta-turn antimicrobial peptide, PST13-RK, on antimicrobial activity and mammalian cell toxicity.

PST13-RK (KKKFPWWWPFKKK-NH(2)) is an improved derivative of tritrpticin adopting a beta-turn structure. In order to investigate the effect of dimerization of PST13-RK on antimicrobial activity and mammalian cell toxicity, we designed and synthesized its Cys- and Lys-linked dimers. The dimerization of PST13-RK resulted in a 2-4 fold decreased antimicrobial activity against Gram-positive and Gram-negative bacteria. However, the dimers showed a large increase in mammalian cell toxicity against mouse NIH-3T3, human MDA-MB-361, and human A549 cells. These results suggested that PST13-RK is active as a monomer to bacterial cells but as an oligomer to mammalian cells. Since the dimeric PST13-RK is much more effective against the cancer cells than the monomer, it might be an attractive candidate for anticancer chemotherapeutic drugs.

Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure-activity studies.

Redirecting the splicing machinery through the hybridization of high affinity, RNase H- incompetent oligonucleotide analogs such as phosphoramidate morpholino oligonucleotides (PMO) might lead to important clinical applications. Chemical conjugation of PMO to arginine-rich cell penetrating peptides (CPP) such as (R-Ahx-R)(4) (with Ahx standing for 6-aminohexanoic acid) leads to sequence-specific splicing correction in the absence of endosomolytic agents in cell culture at variance with most conventional CPPs. Importantly, (R-Ahx-R)(4)-PMO conjugates are effective in mouse models of various viral infections and Duchenne muscular dystrophy. Unfortunately, active doses in some applications might be close to cytotoxic ones thus presenting challenge for systemic administration of the conjugates in those clinical settings. Structure-activity relationship studies have thus been undertaken to unravel CPP structural features important for the efficient nuclear delivery of the conjugated PMO and limiting steps in their internalization pathway. Affinity for heparin (taken as a model heparan sulfate), hydrophobicity, cellular uptake, intracellular distribution and splicing correction have been monitored. Spacing between the charges, hydrophobicity of the linker between the Arg-groups and Arg-stereochemistry influence splicing correction efficiency. A significant correlation between splicing correction efficiency, affinity for heparin and ability to destabilize model synthetic vesicles has been observed but no correlation with cellular uptake has been found. Efforts will have to focus on endosomal escape since it appears to remain the limiting factor for the delivery of these splice-redirecting ON analogs.