Membrane Fusion Peptides Research Articles

A strategy for producing isotopically labeled peptides with antimicrobial activity or with short in vivo lifetime in Escherichia coli

AbstractEngineered Escherichia coli (E. coli) strains have been widely used to produce isotopically labeled peptides for NMR characterization on their structures and interactions. However, production of antimicrobial peptides (AMPs) by E. coli is still challenging, because AMPs are toxic to E. coli host and would lead to cell death after induction. On the other hand, expression of short peptides in E. coli host often encounter problems of the short in vivo lifetime of the peptides, which were rapidly degraded by endogenous enzymes during expression and purification steps. This report presents a practical method for overcoming these bottlenecks to enable E. coli to express AMPs and peptides that have short in vivo lifetime. This design uses the fusion of thioredoxin tags at both the N‐ and C‐termini of the target peptides. The steric effect of the large soluble tags at both ends of the peptide reduces peptide accessibility, thereby enhancing their in vivo stability and eliminating the toxicity associated with AMPs. The approach was validated using an AMP A3K/L7K‐LAH4 (K3K7) and a membrane fusion peptide (FP), which is a segment of the spike protein of SARS‐CoV‐2 and functions in fusing viral membranes and host cell membranes. Fusion expression of K3K7 with a thioredoxin tag only at the N‐terminal resulted in high toxicity to the host cells, leading to impaired cell growth and a failure to obtain expressed fusion protein. In contrast, the fusion proteins from both termini were successfully expressed and purified. In the case of the FP, the fusion of thioredoxin at both termini significantly enhanced its stability, protecting it from enzymatic degradation during expression and purification steps. On the contrary, the FP with thioredoxin fused only at the N‐terminal was found to be unstable in E. coli host strains. As stable isotope labeling on peptide is essentially important in NMR‐based structure and interaction studies, we also demonstrated that the developed approach enables efficient 15N labeling for NMR studies. This strategy may also be extended to produce other challenging peptides.

Synthesis of asymmetrically dihydrophobic chain poly(ethylene glycol) lipids for long circulation and membrane fusion

AbstractLiposomes are the predominant drug delivery system widely used in the pharmaceutical industry, but rapid clearance and endosome degradation are the main barriers to improve the delivery efficiency of liposomes, especially for protein or genes. Simulating viral membrane fusion peptides, four asymmetrically dihydrophobic chain polyethylene glycol (PEG) lipids with long circulating and membrane fusion properties have been designed and synthesized with a high yield. The structure of synthetic PEG lipids is characterized by 1H NMR and mass spectrometry. The critical micelle concentrations of PEG lipids are lower by one or two orders of magnitude compared with traditional surfactants such as SDBS or SDS, which show excellent self‐assembly performance of lipids. The size and morphology of liposomes constituted with neutral lipid DOPC and PEG lipid are characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). In the conditions of 50% ethanol and 5–25 mmol L−1 Ca2+, the PEG neutral lipid complexes encapsulated pEGFP are almost neutral with a diameter below 300 nm.

Bicontinuous inverted cubic phase stabilization as an index of antimicrobial and membrane fusion peptide activity

Some antimicrobial peptides (AMPs) and membrane fusion-catalyzing peptides (FPs) stabilize bicontinuous inverted cubic (QII) phases. Previous authors proposed a topological rationale: since AMP-induced pores, fusion intermediates, and QII phases all have negative Gaussian curvature (NGC), peptides which produce NGC in one structure also do it in another. This assumes that peptides change the curvature energy of the lipid membranes. Here I test this with a Helfrich curvature energy model. First, experimentally, I show that lipid systems often used to study peptide NGC have NGC without peptides at higher temperatures. To determine the net effect of an AMP on NGC, the equilibrium phase behavior of the host lipids must be determined. Second, the model shows that AMPs must make large changes in the curvature energy to stabilize AMP-induced pores. Peptide-induced changes in elastic constants affect pores and QII phase differently. Changes in spontaneous curvature affect them in opposite ways. The observed correlation between QII phase stabilization and AMP activity doesn't show that AMPs act by lowering pore curvature energy. A different rationale is proposed. In theory, AMPs could simultaneously stabilize QII phase and pores by drastically changing two particular elastic constants. This could be tested by measuring AMP effects on the individual constants. I propose experiments to do that. Unlike AMPs, FPs must make only small changes in the curvature energy to catalyze fusion. It they act in this way, their fusion activity should correlate with their ability to stabilize QII phases.

Membranotropic and biological activities of the membrane fusion peptides from SARS-CoV spike glycoprotein: The importance of the complete internal fusion peptide domain.

Fusion peptides (FP) are prominent hydrophobic segments of viral fusion proteins that play critical roles in viral entry. FPs interact with and insert into the host lipid membranes, triggering conformational changes in the viral protein that leads to the viral-cell fusion. Multiple membrane-active domains from the severe acute respiratory syndrome (SARS) coronavirus (CoV) spike protein have been reported to act as the functional fusion peptide such as the peptide sequence located between the S1/S2 and S2’ cleavage sites (FP1), the S2’-adjacent fusion peptide domain (FP2), and the internal FP sequence (cIFP). Using a combined biophysical approach, we demonstrated that the α-helical coiled-coil-forming internal cIFP displayed the highest membrane fusion and permeabilizing activities along with membrane ordering effect in phosphatidylcholine (PC)/phosphatidylglycerol (PG) unilamellar vesicles compared to the other two N-proximal fusion peptide counterparts. While the FP1 sequence displayed intermediate membranotropic activities, the well-conserved FP2 peptide was substantially less effective in promoting fusion, leakage, and membrane ordering in PC/PG model membranes. Furthermore, Ca2+ did not enhance the FP2-induced lipid mixing activity in PC/phosphatidylserine/cholesterol lipid membranes, despite its strong erythrocyte membrane perturbation. Nonetheless, we found that the three putative SARS-CoV membrane-active fusion peptide sequences here studied altered the physical properties of model and erythrocyte membranes to different extents. The importance of the distinct membranotropic and biological activities of all SARS-CoV fusion peptide domains and the pronounced effect of the internal fusion peptide sequence to the whole spike-mediated membrane fusion process are discussed.

Enhanced nuclear delivery of anti-cancer drugs using micelles containing releasable membrane fusion peptide and nuclear-targeting retinoic acid.

Biodegradable cross-linked N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer micelles can improve the accumulation of drug cargo in tumors by prolonging their circulation time. However, drug delivery can still be ineffective because of intracellular degradation in lysosomes and poor delivery to the nucleus. In this work, we prepared a novel micelle by grafting the hydrophobic HA2 membrane fusion peptide onto hydrophilic HPMA copolymers via a linker that would be cleaved in lysosomes, allowing the HA2 peptide to be released and disrupt lysosome membranes. In addition, we conjugated the drug cargo (H1 peptide) to nucleus-targeting all-trans retinoic acid, and then encapsulated the conjugates into micelles. The drug-loaded micelles efficiently escaped lysosomes and targeted the nucleus in MCF-7 breast cancer cells in culture. They also strongly inhibited tumor growth in mice bearing MCF-7 tumor xenografts, without causing appreciable systemic toxicity. Removing the retinoic acid or preventing the cleavage of HA2 resulted in extremely inefficient lysosomal escape and nuclear delivery, translating into low anti-cancer efficacy in vitro and in vivo. These results suggest that micelle modifications to evade lysosomes and target the nucleus can improve the efficacy of anti-cancer drugs. Our results further suggest that the ability to escape lysosomes improves the nuclear distribution of drug cargos more than the addition of the nuclear-targeting retinoic acid.

Effect of Dengue Fusion Peptide in Langmuir Monolayers

Membrane fusion peptides are part of Dengue virus system, serving as a gateway to the virus access into the cell. Studies on the mechanisms of action of these peptides to improve protocols for the treatment and achievement of new drugs to combat the dissemination of the virus are increasingly necessary. The Langmuir technique is very useful to study peptide-lipid interactions. Indeed, the utilization of lipid monolayers allows controlling several physical parameters such as lipid composition, surface pressure and many other parameters of interest. Here we study the interaction of the Dengue Flavivirus fusion peptide (FLAg) with Langmuir monolayers composed of different types of lipids, including POPG, (palmitoyl-oleoyl-phosphatydyl-glycerol). In order to assess peptide maximum insertion pressure (MIP) into the Langmuir model membrane, kinetic measurements were performed. The kinetics of FLAg binding onto the phospholipid monolayer was monitored until the equilibrium surface pressure (Πe) was reached. The MIP was determined by injecting FLAg at different initial surface pressures (Πi) of the lipid monolayers. To investigate the behavior of FLAg in Langmuir monolayers with different lipid composition, we also used in situ infrared spectroscopy (PM-IRRAS). Preliminary results showed an increase in surface pressure during adsorption kinetics experiments with POPG monolayers, revealing an electrostatic affinity that evidences the role of the membrane charge in the fusion process. Furthermore, surface activity of the peptide solution used as the subphase in the absence of lipid monolayers was also measured. PM-IRRAS measurements reveal how the peptide incorporates at the interface of POPG monolayers, showing changes in the spectra, besides indicating characteristic due to the presence of FLAg. Altogether, these data clearly demonstrate that useful information on the binding FLAg to membranes can be obtained by performing measurements of their monolayer binding parameters.

Effect of Dengue Fusion Peptide in Anionic Lipid Bilayers

Studies involving peptide-induced membrane fusion are extremely important to better understanding the mechanisms underlying virus entry in cells. Particularly, membrane fusion peptides are part of Dengue virus system serving as a gateway to the virus access into the cell. Here we study the interaction of the Dengue Flavivirus fusion peptide (FLAg) with model membranes composed of anionic phospholipid, POPG, (palmitoyl-oleoyl-phosphatydil-glycerol). We used large (LUV) and giant (GUV) unillamelar vesicles as biomimetic membranes systems.

Biophysical and functional assays for viral membrane fusion peptides

Membrane fusion is a protein catalyzed biophysical reaction that involves the simultaneous intermixing of two phospholipid bilayers and of the aqueous compartments bound by their respective bilayers. In the case of enveloped virus fusogens, short hydrophobic or amphipathic fusion peptides that are components of the larger fusion complex are essential for the membrane merger event. The process of cell–cell membrane fusion and syncytium formation induced by the nonenveloped fusogenic orthoreoviruses is driven by the Fusion-Associated Small Transmembrane (FAST) proteins, which are similarly dependent on the action of fusion peptides. In this article, we describe some simple methods for the biophysical characterization of viral membrane fusion peptides. Liposomes serve as an ideal model system for characterizing peptide–membrane interactions because their size, shape and composition can be readily manipulated. We present details of fluorescence assays used to elucidate the kinetics of membrane fusion as well as complimentary assays used to characterize peptide-induced liposome binding and aggregation.

The fusion peptide inhibits the growth of nude mice colon xenograft tumor

Objective To observe whether the through membrane fusion peptide TAT-N24 can inhibit the growth of nude mice colon xenograft tumors. Methods To study the effect of TAT-N24, or TAT-N24m (negative control) on tumor growth,we constructed the nude mice colon xenograft tumors model, injection TAT-N24 and TAT-N24m in veins, comparing tumor weight change, BrdU incorporation was used to detect the effect of TAT-N24 on cell DNA synthesis,in vivo experiments proof the effect of TAT-N24 on anti-tumor. Results TAT-N24 and negative control peptide TAT-N24m can be detected by immunohistochemistry through HAtag positive expression, suggesting that the fusion peptide can effectively enter into tumor cells. TAT-N24 group slow down tumor growth significantly, tumor weight in each group were: average weight of tumors in each group, Blank Group: ( 0. 34 ± 0. 09 ) g; TAT-N24m group: ( 0. 27 ± 0. 06 ) g; TAT-N24 Group: (0. 12 ± 0. 02 ) g, suggesting that TAT-N24 can inhibit the growth of HT29 cells in nude mice xenogaraft tumors (P ＜ 0. 05 ). BrdU incorporation shows that BrdU-positive cells of TAT-N24 group were significantly lower than the control group. Conclusion The fusion peptide TAT-N24 can effectively inhibit the growth of nude mice colon xenograft tumor and the DNA synthesis in the tumor cells. Key words: Tumor; Colon Cancer; BrdU; PI3K

Characterization of the Putative Membrane Fusion Peptides in the Envelope Proteins of Human Hepatitis B Virus

Envelope proteins of virus contain a segment of hydrophobic amino acids, called as fusion peptide, which triggers membrane fusion by insertion into membrane and perturbation of lipid bilayer structure. Potential fusion peptide sequences have been identified in the middle of L or M proteins or at the N-terminus of S protein in the envelope of human hepatitis B virus (HBV). Two 16-mer peptides representing the N-terminal fusion peptide of the S protein and the internal fusion peptide in L protein were synthesized, and their membrane disrupting activities were characterized. The internal fusion peptide in L protein showed higher activity of liposome leakage and hemolysis of human red blood cells than the N-terminal fusion peptide of S protein. Also, the membrane disrupting activity of the extracellular domain of L protein significantly increased when the internal fusion peptide region was exposed to N-terminus by the treatment of V8 protease. These results indicate that the internal fusion peptide region of L protein could activate membrane fusion when it is exposed by proteolysis.

2P260 膜融合ペプチドにより引き起こされるリポソームと脂質平面膜の相互作用の全反射蛍光顕微鏡観察(生体膜・人工膜(構造・物性),ポスター発表,第45回日本生物物理学会年会)

2P260 膜融合ペプチドにより引き起こされるリポソームと脂質平面膜の相互作用の全反射蛍光顕微鏡観察(生体膜・人工膜(構造・物性),ポスター発表,第45回日本生物物理学会年会)

Precisely controlled intracellular delivery of DNA-peptide conjugates

Precisely controlled intracellular delivery of oligonucleotides was achieved by conjugation with signal peptides and amphiphilic designed peptides. Localization in the cellular nucleus was accelerated by nuclear localizing signals derived from naturally occurring viral proteins and arginine rich designed peptides, and cytoplasmic localization was enhanced by nuclear export signals and membrane fusion peptides.

Structure and function of membrane fusion peptides.

Membrane fusion peptides are highly conserved hydrophobic domains of fusion proteins that insert into membranes during membrane fusion. Recent success with solving the structures of the influenza hemagglutinin fusion peptide and some critical mutants of this peptide in membrane environments at high resolution has led to a new understanding of the mechanism of membrane fusion. This review highlights the structures that have been solved and summarizes recent thermodynamic and spectroscopic studies on the interactions of this interesting class of peptides with lipid bilayers.

Editorial: Current trends in peptide science

This “Current Trends in Peptide Science” issue features papers broadly based on the diversity of biologically-important interactions of peptides and proteins with cellular membranes. In the first paper, Unson focuses on the seven-helix transmembrane receptor for glucagon, a 29-residue peptide hormone that functions to counterbalance the effects of insulin in blood glucose levels. She describes progress toward the elucidation of molecular determinants of ligand specificity by the receptor, itself a member of the G-protein coupled receptor (GPCR) family, and discusses models which have been proposed for the specificity of binding of these so-called Family B receptors to a series of highly homologous peptide ligands. Noting the increasing resistance of bacteria to conventional antibiotics, Shai then presents an in-depth discussion of a new generation of antibiotic peptides. Because these peptides are active across a wide range of lengths and sequences, he presents evidence for the “carpet model” of peptide antibiotic action—a generalized mechanism for bacterial membrane binding and penetration which may be coupled in some instances with eventual targeting of the peptides to the cytoplasmic region of the organism. In a related theme, Tamm presents a review of membrane fusion peptides that are constituted of highly conserved hydrophobic segments of fusion proteins; these peptides which mediate processes requiring two cellular membranes to fuse into one. Focusing on influenza hemagglutinin fusion peptides, Tamm describes biophysical approaches—including NMR spectroscopy. ATR infrared techniques, and differential scanning calorimetry—to deduction of the structures and interactions of these peptides with phospholipid bilayers. McCafferty and his co-workers conclude this issue with a discussion of the wide-ranging properties of the peptide antibiotic ramaplanin family—compounds structurally related to cell wall-active lipodepsipeptides—which may find clinical use against vancomycin-resistant and methicillin-resistant pathogens. The collection of papers in this issue truly reflects the important advances in our understanding of peptide/protein interactions with membranes—whether involving receptor-mediated recognition, surface binding, membrane penetration/fusion, or mechanisms of antibiotic action—as well as highlighting the momentum for continuing research in these emerging fields.

A host-guest system to study structure-function relationships of membrane fusion peptides.

We designed a host-guest fusion peptide system, which is completely soluble in water and has a high affinity for biological and lipid model membranes. The guest sequences are those of the fusion peptides of influenza hemagglutinin, which are solubilized by a highly charged unstructured C-terminal host sequence. These peptides partition to the surface of negatively charged liposomes or erythrocytes and elicit membrane fusion or hemolysis. They undergo a conformational change from random coil to an obliquely inserted ( approximately 33 degrees from the surface) alpha-helix on binding to model membranes. Partition coefficients for membrane insertion were measured for influenza fusion peptides of increasing lengths (n = 8, 13, 16, and 20). The hydrophobic contribution to the free energy of binding of the 20-residue fusion peptide at pH 5.0 is -7.6 kcal/mol (1 cal = 4.18 J). This energy is sufficient to stabilize a "stalk" intermediate if a typical number of fusion peptides assemble at the site of membrane fusion. The fusion activity of the fusion peptides increases with each increment in length, and this increase strictly correlates with the hydrophobic binding energy and the angle of insertion.

Mobile Phospholipid Bilayers Supported on a Polyion/Alkylthiol Layer Pair

The formation of phospholipid bilayers on a polymer/alkylthiol layer pair was investigated by surface plasmon resonance (SPR). The organic layer pair between the lipid membrane and the solid gold surface consisted of a self-assembled monolayer of 11-mercaptoundecanoic acid (MUA) on a gold surface followed by a thin layer of hydrated cationic poly(diallyldimethylammonium chloride) (PDDA). The lipid layers were formed by vesicular fusion of small unilamellar vesicles of an anionic lipid 1-stearoyl-2-oleoylphosphatidylserine (SOPS), a zwitterionic lipid 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and mixtures of these two lipids with different compositions. In the case of pure SOPS and lipid mixtures with a POPC composition below 25%, the lipid layer thickness was approximately that of a single bilayer, while multilayers tended to build up with higher concentrations of POPC. The electrostatic interaction of the cationic PDDA with the negatively charged lipid membrane is most probably the driving force for the adsorption of a single bilayer. Fluorescence recovery after photobleaching (FRAP) experiments showed that single bilayers supported on the PDDA/alkylthiol layer pair were mobile at room temperature with lateral diffusivities of approximately (1−2) × 10-9 cm2/s. A membrane fusion peptide wt-20 of the influenza virus was bound to the supported bilayers as detected by SPR. This suggests the potential of this model membrane system for use as a biosensor.

Effects on mollicutes (wall-less bacteria) of synthetic peptides comprising a signal peptide or a membrane fusion peptide, and a nuclear localization sequence (NLS) – a comparison with melittin

In order to investigate the effect of primary amphipathic peptides on mollicutes (wall-less bacteria), we have synthesised five molecules (P1, P2, P3, JM123, and JM133) comprising a 16 to 18-residue hydrophobic sequence and the nuclear localization sequence (NLS) PKKKRKV of simian virus 40 large-T antigen, C-terminated by a cysteamide group. The hydrophobic cluster was in P1 the signal sequence of the heavy chain of Caiman crocodilus immunoglobulin G and in JM123 the fusion peptide of human immunodeficiency virus 1 glycoprotein gp41 in which phenylalanine 7 was replaced by a tryptophan residue. The homologues P2, P3, and JM133 were obtained by slight alterations of these sequences. Circular dichroism spectroscopy revealed that, in liposomes, P-series peptides were mainly under the form of β-sheets whereas JM-series peptides displayed a high proportion of turns. These peptides proved to be bactericidal for some mollicutes, notably Acholeplasma laidlawii, but were much less potent than melittin. Furthermore, their antibiotic activity was independent of the average thickness of the plasma membrane hydrophobic core whilst that of melittin was inversely related to the thickness. Melittin and the synthetic peptides abolished spiroplasma cell motility and helicity, but only melittin and P-series peptides split the cells into globular forms displaying an average diameter of ca. 1 μm. In contrast to melittin, the synthetic peptides agglutinated spiroplasmas, suggesting that their polycationic NLS was exposed on the cell surface. P-series peptides decreased, though less efficiently than melittin, A. laidlawii and Spiroplasma melliferum membrane potential (Δ Ψ) and transmembrane pH gradient (ΔpH), at concentrations much lower than their minimal inhibitory concentrations whilst JM-series peptides had no effect on Δ Ψ and ΔpH in the same conditions. Actually, the bactericidal activity of these peptides towards mollicutes was proportional to their ability to collapse the electrochemical transmembrane potential.

Viruses

The structures of the components of large and complex viruses, determined over the past year, have demonstrated the great variation in the ways in which viruses achieve their goals. The structure of the bluetongue virus coat protein provides clues as to how a T = 13 particle is assembled and the structure of the tick-borne encephalitis envelope protein suggests a new way of exposing a membrane fusion peptide at the right moment.