Protein and Peptide Letters - Volume 16, Issue 7, 2009

I am very pleased to offer to the readers of Protein and Peptide Letters this special issue entitled “Developments in membrane fusion” highlighting the latest findings in the field. The aim of this issue is not to provide an exhaustive collection of data on membrane fusion but rather to present an updated, hopefully general, overview on this process and provide ideas and information that could contribute to the reader's own research. Membrane fusion has attracted great interest among scientists in recent years. This process is fundamental to health and disease: it occurs at fertilization, is needed for hormones release into the bloodstream and during development, but it is also the mechanism used by enveloped viruses to enter cells or carcinogenesis. In the last few years, great strides have been made in our understanding of the molecular machinery and mechanism of membrane fusion. Fusion machines are adapted to fit the needs of different reactions but operate by similar principles in order to achieve merging of the bilayers. In spite of the ubiquity of membrane fusion, scientists are still trying to solve the mystery of how different molecules drive vesicles fusion. Understanding the details of membrane fusion may help scientists to find the appropriate conditions for preventing viruses from fusing to and thereby infecting human cells and could also lead to the design of systems in which a drug, enclosed in a membrane known to fuse with specific cells in our body, may be delivered or to improve gene therapy. A number of research groups in the world are focused on cellular and biophysical aspects of fusion and are directed at understanding the protein components and/or membrane interactions that are necessary to facilitate and trigger fusion. These groups are making leading contributions to understanding the membrane perturbations and protein interactions that promote fusion as well as the cellular machinery that directs fusion. This special issue includes several papers describing the know-how on membrane fusion.

Life processes are governed at the chemical level, and therefore knowledge of how single molecules interact, provides a fundamental understanding of nature. The molecular mechanism of membrane fusion essential to vital cellular activities such as intracellular transport, hormone secretion, enzyme release, or neurotransmission, involve the assembly and disassembly of a specialized set of proteins present in opposing bilayers. Target membrane proteins at the cell plasma membrane SNAP-25 and syntaxin termed t-SNAREs, and secretory vesicle-associated protein VAMP or v-SNARE, are part of the conserved protein complex involved in fusion of opposing membranes. It has been demonstrated that in the presence of Ca2+, t-SNAREs and v-SNARE in opposing bilayers interact and self-assemble in a circular pattern, to form conducting channels. Such self-assembly of t-/v-SNAREs in a ring conformation occurs only when the respective SNAREs are in association with membrane. X-ray diffraction measurements further demonstrate that t-SNAREs in the target membrane and v-SNARE in the vesicle membrane overcome repulsive forces to bring opposing membranes close to within a distance of 2.8 Å. Studies suggest that calcium bridging of the opposing bilayers, lead to release of water from hydrated Ca2+ ions as well as the loosely coordinated water at PO-lipid head groups, leading to membrane destabilization and fusion. The t-/v-SNARE is a tight complex, who's disassembly requires an ATPase called NSF, which functions as a right-handed molecular motor.

Class I fusion glycoproteins of viruses are involved in the fusion between viral envelope and cell membrane. A region located in the N-terminal domain of these glycoproteins, called the fusion peptide, is essential for fusion. Fusion peptides are able to induce by themselves in vitro membrane fusion. In this paper, we review the properties of those peptides related to their fusogenicity, in particular the correlation existing between their ability to insert obliquely in membranes and fusogenicity. This relation notably allows predicting successfully the minimal region of some fusion peptides sufficient to induce significant in vitro fusion. The notion of obliquity and fusogenicity is discussed in terms of the existing proposed mechanisms for viral fusion.

Interaction of fusogenic or membrane-perturbing peptides with lipid bilayers often involves drastic rearrangements of the membrane structure, with redistribution of lipids, inducement of bilayer curvature, or formation of nonbilayer or multibilayer structures. Fluorescence (or Forster) Resonance Energy Transfer (FRET) is a photophysical technique which has an acute sensitivity to distances in the nanometer range, and, as such, is particularly suited to probe alterations in membrane organization in this length scale. This article reviews methods and selected applications of FRET in this field, from the now classic (fusion induced) lipid-mixing assay to examples where kinetic modeling of FRET enables the recovery of topological information.

Antimicrobial peptides (AMP's) are promising compounds in the battle against antibiotic resistant pathogens. Many AMP's function by interacting with the bacterial membrane and selectively permeabilizing it. Improvements are desired in the potency and the in vivo stability of the AMP's. Both aspects have been approached by the preparation of multivalent versions of AMP's that contain several copies of the peptide attached to a scaffold or core molecule. Both short and long sequences have been used and in selected cases major increases in antibacterial activity, membrane permeabilization potency and in vivo stability have been obtained.

Similarly to antimicrobial peptides (AMPs), viral fusion peptides (FPs) are membrane-active peptides. This minireview emphasizes the common properties of AMPs and FPs with a special focus on the intrinsic flexibility and structural adaptability of these peptides that are responsible for different mode of interaction with the membrane bilayers. The potential use of AMPs as multifunctional drugs with both antibacterial and antiviral properties is discussed.

Membrane fusion and fission are two key processes that occur during the replication of enveloped viruses, namely access to the interior of the host-cell (entry, which requires fusion of the viral envelope with the target cell envelope) and dissemination of viral progeny after replication (egress, which involves budding and fission). These dynamic processes are mediated by specialized proteins that modify and bend the lipid bilayer transiently and locally. This review focuses on fusion and fission reactions and on the hypothetical shared mechanism that generates their driving force.

The mechanism by which herpesviruses fuse with cellular membranes to permit virus entry is still relatively poorly understood. This process is proving difficult to unravel, largely due to the fact that multiple viral envelope proteins appear to function in concert to mediate the fusion event. For Herpes Simplex Virus Type 1 (HSV1), glycoproteins B, D and the gHL heterodimer are all required for fusion, and gHL counterparts are involved in the fusion process of all other members of the herpesvirus family. An understanding of the functional domains of gH that are critical for fusion may offer the possibility of designing specific peptide inhibitors of virus entry, and recent progress has highlighted the potential usefulness of this approach. This review discusses these advances and outlines some of the similarities and differences between gH homologues of the different members of this diverse family of viruses.

Fusion of the influenza virus envelope with the endosomal membrane of host cells is mediated by the hemagglutinin glycoprotein (HA). The most conserved region of HA is at the N-terminus of the HA2 subunit, a relatively hydrophobic sequence of amino acids referred to as the fusion peptide. This domain is critical both for setting the trigger for fusion and for destabilizing target membranes during the fusion process. The “trigger” is set by cleavage of the HA precursor polypeptide, when the newly-generated HA2 N-terminal fusion peptide positions itself into the trimer interior and makes contacts with ionizable residues to generate a fusion competent neutral pH structure. This essentially “primes” the HA such that subsequent acidification of the endosomal environment can induce the irreversible conformational changes that result in membrane fusion. A key component of these acid-induced structural rearrangements involves the extrusion of the fusion peptide from its buried position and its relocation to interact with the target membrane. The role of the fusion peptide for both priming the neutral pH structure and interacting with cellular membranes during the fusion process is discussed.

Membrane fusion is an essential step in the entry of enveloped viruses into their host cells, what makes it a potentially attractive target for viral inactivation approaches. Fusion is mediated by viral surface glycoproteins that undergo conformational changes triggered by interaction with specific cellular receptors or by the exposition to low pH of endossomal medium. Here we review how several studies on the structural rearrangements of vesicular stomatitis virus (VSV) glycoprotein G during cellular recognition and fusion led us to propose a crucial role of the protonation of His residues for G protein activity. Moreover, we demonstrated that using diethylpyrocarbonate (DEPC), a histidine-modifying compound, it was possible to abolish viral infectivity and pathogenicity in mice, and to elicit neutralizing antibodies that confer protection in these animals against challenge using lethal doses of the virus. The presence of conserved His residues in a wide range of viral fusion proteins and the use of DEPC as a more general means for vaccine development will be also discussed.

Enveloped animal viruses fuse their membrane with a host cell membrane in order to deliver their genome into the cytoplasm of the cell and thus initiating infection. This crucial step is mediated by virally encoded transmembrane proteins that, following an appropriate triggering, insert their fusion peptides into the target membrane and, through a cascade of conformational changes, drive the merging of the two apposing membranes. The battle against viruses is ongoing with the constant threat of viruses developing resistance to present drugs and emerging viruses, therefore there is a continuous challenge to improve our defence strategies. Entry inhibitors are currently in development for diverse human and animal viral pathogens, and advances in our understanding on how viral entry proteins undergo conformational changes that lead to entry offer a huge potential for the development of novel therapeutics. This review describes recent advances on viralmediated fusion mechanisms concentrating on the development of peptidic inhibitors of membrane fusion.

Since 1999 we have developed three approaches to quantifying each amino acid in a protein as well as a protein in whole based on random mechanisms. With our approaches, we can reliably describe the evolution of a protein family, for example, the hemagglutinins from influenza A viruses along the time course in a 2-dimensional graph, and then we use the fast Fourier transform to find the mutation periodicity in order to time the mutation. In this study, we realize that the changes in quantified randomness in a hemagglutinin family over time is the difference between randomness associated with mutant amino acids and randomness associated with original amino acids. This is a standard mass-balance relationship, by which we can build a differential equation for a hemagglutinin family or a system of differential equations for all hemagglutinins in the family. In this context, the randomness defined by us actually is the entropy, thus we have a general model to describe the evolution, namely, the evolution is the exchange of entropy between protein family and environment through mutations quantified using our approaches.

A series of 30 tripeptides were synthesized and tested as novel 5-HT4 receptor ligands. Receptor binding assays showed that a subset of compounds had reasonable potency relative to the agonists serotonin and 5-methoxytryptamine. Structure-activity analyses and molecular docking have highlighted avenues for further synthetic work.

The N-terminal 25 residue segment of human surfactant protein B (SP-B1-25) was synthesised in 26% yield by manual Fmoc solid-phase peptide synthesis (Fmoc SPPS) using low-loading Fmoc-Gly-Wang resin. Substantial oxidation of Met21 occurred during the synthesis, and the addition of Bu4NBr to a TFA/water/EDT/TIS cleavage cocktail enabled facile reduction of Met(O)21-SP-B1-25 to SP-B1-25. The methods described herein are generally applicable to the Fmoc SPPS of difficult sequences containing methionine.

The mechanism(s) by which hepatitis C virus (HCV) enters and infects cells remains unknown. Identifying the HCV fusion peptide(s) and understanding the early stages of infection may provide new opportunities for improved antiviral therapy. The HCV envelope glycoprotein E2 is thought to be a class II fusion protein. Class II fusion proteins are exemplified by the E protein of the tick-borne encephalitis virus (TBEV) and the E1 protein of the Semliki Forest virus (SFV). Analysis of the hydrophobicity profiles of four HCV E2 envelope glycoproteins revealed a region with a conserved three-pronged pattern of hydrophobicity, termed the tridentate (TD) region. The primary sequence of the TD region is highly conserved in all 490 HCV strains currently reported. The known fusion peptide loops of TBEV and SFV share the characteristic TD region hydrophobicity profile and significant sequence conservation in the TD region was identified in the E and E1 glycoproteins of members of the Flaviviridae and Togaviridae families, respectively. The HCV TD region peptides have membranotropic activity; in molecular dynamics (MD) simulations, the HCV TD region peptides insert into in a biomimetic bilayer in a similar manner to the TBEV fusion peptide and the peptides induce effective mixing of lipid membranes in a liposome fusion assay. Together these results indicate that the highly conserved TD region of the HCV E2 protein is a fusion peptide candidate and may be an important factor in the class II fusion mechanism.

Nuclear receptors are involved in multiple cellular signaling pathways that affect and regulate processes. Because of their physiology and pathophysiology significance, classification of nuclear receptors is essential for the proper understanding of their functions. Bhasin and Raghava have shown that the subfamilies of nuclear receptors are closely correlated with their amino acid composition and dipeptide composition [29]. They characterized each protein by a 400 dimensional feature vector. However, using high dimensional feature vectors for characterization of protein sequences will increase the computational cost as well as the risk of overfitting. Therefore, using only those features that are most relevant to the present task might improve the prediction system, and might also provide us with some biologically useful knowledge. In this paper a feature selection approach was proposed to identify relevant features and a prediction engine of support vector machines was developed to estimate the prediction accuracy of classification using the selected features. A reduced subset containing 30 features was accepted to characterize the protein sequences in view of its good discriminative power towards the classes, in which 18 are of amino acid composition and 12 are of dipeptide composition. This reduced feature subset resulted in an overall accuracy of 98.9% in a 5-fold cross-validation test, higher than 88.7% of amino acid composition based method and almost as high as 99.3% of dipeptide composition based method. Moreover, an overall accuracy of 93.7% was reached when it was evaluated on a blind data set of 63 nuclear receptors. On the other hand, an overall accuracy of 96.1% and 95.2% based on the reduced 12 dipeptide compositions was observed simultaneously in the 5-fold cross-validation test and the blind data set test, respectively. These results demonstrate the effectiveness of the present method.

Familial mediterranean fever (FMF) is a familial disease characterized by recurrent episodes of febrile serositis, peritonitis, arthritis and pleuritis. Many studies have been performed is an attempt to understand the basis of the inflammatory attacts in FMF. Ghrelin, a recently described orexigene peptide is predominantly produced by stomach. Ghrelin also exerts multiple regulatory effects on immune system. It has reported that grelin has anti-inflammatory effects. There is currently no published evidence demonstrating a role for anti-inflammatory effects of ghrelin in FMF. For this reason, we investigated the role of plasma ghrelin levels in patients with FMF. Thirty seven patients with FMF and 10 healthy controls (5 female, 5 male; mean age 35.4 ± 5.6 years) were enrolled in this study. Twenty-one patients were in active stage (10 female, 11 male, mean age; 31.0 ± 5.4 years, mean disease duration 7.2 ± 3.3 years) and 16 patients were in inactive stage (7 female,9 male, mean age; 33.0 ± 6.0 years, mean disease duration; 8.7 ± 3.2 years). Plasma ghrelin levels were determined by EIA. The mean plasma ghrelin levels were 158.4 ± 52.9 pg/ml in patients with FMF and 56.7 ± 7.5 pg/ml in healthy controls. The mean plasma ghrelin levels were 190.5 ±49.4 pg/ml in the active patients and 116.2 ± 11.7 pg/ml in the inactive patients. Plasma ghrelin levels were significantly high in patients with FMF compared to healthy controls (p<0.001). Plasma ghrelin levels were significantly high in the active patients compared to in the inactive patients and healthy controls (p<0.001 and p<0.001 respectively). There was significantly difference between in active and inactive patients with FMF (p<0.001). As a results; Plasma ghrelin levels were high both in active and inactive patients with FMF. It is showed that ghrelin may play significant role of the pathogenesis of FMF.

The successful folding of a recombinant protein after expression and purification is essential for structural, biochemical and vaccination studies. Toxoplasma gondii recombinant GRA1 protein is a promising vaccine candidate against toxoplasmosis. In the present study, the folding of recombinant GRA1 protein has been evaluated by web based bioinformatics tools that predict protein folding. Subsequently, trypsin digestion, which is a simple indication of proper protein folding, has been used to determine whether recombinant GRA1 protein is likely to be folded. The results indicate that the recombinant GRA1 protein is predicted to be folded by most of the web based bioinformatics predictors. Moreover, in protease digestion experiments, the recombinant GRA1, which was purified to homogeneity without the use of denaturants, gives rise to a discrete band pattern that is indicative of a folded protein. Together, the results suggest that recombinant GRA1 protein is in a folded conformation, suitable for structural, biochemical and vaccination studies.

For chronic viral infections like Hepatitis C, CD8-CTLs have emerged as important protective tools. Hence, isolated dominant epitopes arranged as polytope DNA or peptide vaccines represent a promising approach. However, because of controversial rules governing the polytope construction and epitope processing, proper design and primary analysis of such vaccines are prior to the costly transgenic animal studies. In this study, based on in silico epitope selection, four HLA-A2 (C132, E614 and N1406) and H-2d (E405 and C132) immunodominant CD8-epitopes of HCV were selected. The codon optimized nucleotide sequences of the epitopes were assembled by overlap extension PCR in three different sequential tandems for the best proteasomal cleavage predictions and cloned into pcDNA3.1 vector. In addition, to enhance particulate formation, three other plasmids containing the fusion of polytopes with hepatitis B surface antigen gene (HBsAg) were also constructed. Proper expression of all constructs in transfected Cos-7 cells was verified by RT-PCR, immunofluorescence, Western-blot, ELISA and dot blot techniques. Moreover, particle formation of HBsAg-fused polytopes was manifested by their secretion to the culture media albeit in lesser amounts compared to sole HBsAg protein. Finally, the positive delayed-type hypersensitivity (DTH) response of vaccinated mice indicated the in vivo expression of all constructs and efficient stimulation of immune response, which was stronger for HBsAg fusion constructs. In addition, proper processing of the epitopes was evidenced by the DTH response towards H-2d epitopic peptides. These data provide enough support and merit for the further evaluation of the designed constructs in HLA-A2 transgenic mice.