Inhibition Of Membrane Fusion Research Articles

Stapled peptide-based membrane fusion inhibitors of hepatitis C virus

The strategy of peptide stapling was used to develop new molecules to inhibit the hepatitis C virus infection via disrupting the binding of HCV envelope glycoprotein E2 with human cell surface protein CD81. The peptide sequence was designed based on the large extra-cellular loop of CD81 with known importance in the HCV E2 binding interaction. Our results showed that the stapled peptides exhibited significantly higher α-helicity and proteolytic stability as compared to their linear peptide counterpart. The optimal compound was found to have an EC50 value of ca. 17–39μM against different HCV subtypes and represented a new HCV membrane fusion inhibitor.

English

Adenosine diphosphate-ribosylation factor 1 (ARF1) is a member of guanosine triphosphate (GTP)-binding proteins family associated with Golgi complexes. ARF1 regulated asymmetric cell division in female meiosis in mouse. However, little isknown about its function in bovine oocyte meiosis. In the present study, we examined the localization, expression and functions of ARF1 during bovine oocyte meiotic maturation. Active form ARF1Q71L-Venus showed that ARF1 co-localized with α-tubulin on the spindle from the M I to the M II stage. Inhibition of ARF1 activity by microinjecting mRNA of a dominant negative mutant form of ARF1 (ARF1T31N ) or ARF1 morpholino (ARF1 MO) into the germinal vesicle (GV) oocytes, and treating GV oocytes with brefeldin A (BFA), an inhibitor of Golgi-based membrane fusion led to abnormal spindle assembly and cytokinesis failure. On the contrary, microinjection of mRNA of a positive mutant form of ARF1 (ARF1Q71L) into GV oocytes had no effect on spindle assembly and the oocytes could undergo normal cytokinesis to generate one large egg with one small polar body. From the above, our results suggest that ARF1 plays an essential role in spindle assembly in bovine oocytes. Key words: Adenosine diphosphate-ribosylation factor 1 (ARF1), spindle assembly, oocytes meiosis cytokinesis, bovine.

Synthesized Peptide Inhibitors of HIV-1 gp41-dependent Membrane Fusion

Fusion of viral and cellular membranes is an essential step for HIV-1 infection. This process offers an attractive target for developing antiviral agents. T20 (Enfuvirtide, Fuzeon), a 36-amino acid peptide derived from the C-terminal heptad repeat region of HIV-1 gp41, is the first and only clinically approved HIV-1 fusion inhibitor that being used for treatment of HIV/AIDS patients failed to respond to current antiretroviral drugs. However, T20-resistance can be acquired rather easily in vitro and in vivo. T1249 is considered as a representative of the second generation fusion inhibitors, but its clinical evaluation was halted due to the problem of drug formulation. To overcome these challenges, a number of strategies have been applied to develop the third or next-generation inhibitors with the significantly improved antiviral activity and pharmacokinetic profiles. Promisingly, several peptides are currently under clinical trials, such as Sifuvirtide and VIR-576. Recently, several high resolution crystal structures of HIV-1 fusion inhibitor peptides were reported, which reveal the key residues or motifs underlying their potency against diverse HIV-1 variants. This review highlights the development of the representative peptide inhibitors of HIV-1 fusion toward providing some insights into the future of this class of anti-HIV drugs.

The Current Status and Challenges in the Development of Fusion Inhibitors as Therapeutics for HIV-1 Infection

HIV-1 membrane fusion as a part of the process of viral entry in the target cells is facilitated by gp41 and gp120, which are encoded by Env gene of HIV-1. Based on the structure and the mechanism researches, new treatment options targeting HIV-1 entry process have been proposed. Enfuvirtide, which mimics amino acid sequences of viral envelope glycoprotein gp41, is the first HIV-1 fusion inhibitor approved by FDA. Although it fulfills vital functions by binding to gp41 and abolishing the membrane fusion reaction when used in combination, it could induce drug resistant virus variants. Currently, a number of design and modification schemes have been presented, a large number of prospective fusion peptides have emerged. For these fusion inhibitors, multiple mutations in gp41 have been associated with the loss of susceptibility to agents. This review reported the current developments and innovative designs of HIV-1 membrane fusion inhibitors.

Mechanism for Active Membrane Fusion Triggering by Morbillivirus Attachment Protein

The paramyxovirus entry machinery consists of two glycoproteins that tightly cooperate to achieve membrane fusion for cell entry: the tetrameric attachment protein (HN, H, or G, depending on the paramyxovirus genus) and the trimeric fusion protein (F). Here, we explore whether receptor-induced conformational changes within morbillivirus H proteins promote membrane fusion by a mechanism requiring the active destabilization of prefusion F or by the dissociation of prefusion F from intracellularly preformed glycoprotein complexes. To properly probe F conformations, we identified anti-F monoclonal antibodies (MAbs) that recognize conformation-dependent epitopes. Through heat treatment as a surrogate for H-mediated F triggering, we demonstrate with these MAbs that the morbillivirus F trimer contains a sufficiently high inherent activation energy barrier to maintain the metastable prefusion state even in the absence of H. This notion was further validated by exploring the conformational states of destabilized F mutants and stabilized soluble F variants combined with the use of a membrane fusion inhibitor (3g). Taken together, our findings reveal that the morbillivirus H protein must lower the activation energy barrier of metastable prefusion F for fusion triggering.

Site-Specific PEGylation of HR2 Peptides: Effects of PEG Conjugation Position and Chain Length on HIV-1 Membrane Fusion Inhibition and Proteolytic Degradation

Peptides derived from the HR1 or HR2 regions of the HIV-1 envelope glycoprotein gp41 have been shown to be effective inhibitors to prevent virus-host cell membrane fusion. These peptide drugs, however, suffer from relatively short plasma half-lives and are susceptible to enzymatic degradation. Modification of peptides/proteins with poly(ethylene glycol) (PEG) is a well-established strategy to overcome these limitations. This manuscript presents the results of a systematic study on the influence of the site of PEGylation of HR2-derived peptides, as well as of PEG molecular weight on the biological activity and proteolytic stability of these conjugates. Investigation of the fusion inhibitory efficacy of the conjugates in a model cell-cell based assay revealed a loss in activity for the PEGylated peptides as compared to the wild-type HR2-derived peptide. The loss of activity, however, can be minimized by controlling the site of PEGylation, more specifically, by introducing the PEG chain at one of the more central positions along the non-interacting α-helical surface of the peptides. The proteolytic stability of the PEG-peptide conjugates was assessed in a trypsin-based model assay, which revealed an up to 3.4-fold increase in degradation half-life that may help to compensate for the lower inhibitory efficacy of the PEG-peptide conjugates as compared to the wild-type peptide. The results of this study emphasize the power of site-specific PEGylation to improve the stability of peptide/protein drugs while minimizing adverse effects on biological activity.

Recent Advances in the Development of Small-Molecule Compounds Targeting HIV- 1 gp41 as Membrane Fusion Inhibitors

Over the past few years, remarkable progress has been made in the development of human immunodeficiency virus (HIV) membrane fusion inhibitors. The focus has been on peptide inhibitors, which were developed by mimicking HIV sequences; however, these types of inhibitors generally lack oral bioavailability and are expensive. Therefore, development of small-molecule inhibitors has gained importance and recently progressed. This paper reviews the rapid advancements in the development of small-molecule HIV inhibitors over the last decade. Keywords: Coiled-coil, docking simulation, gp41, heptad repeat, HIV, membrane fusion, quantitative structure-activity relationship, smallmolecule inhibitors, HAART, drug targets, bioavailability

PH-Sensitive Pegylated Cationic Lipid-DNA Complexes for Gene Delivery: Transfection Efficiency and Live Cell Imaging Studies

Gene therapy is a promising new approach for medicine, and able to target a wide variety of diseases. [1]. Cationic liposome (CL)-DNA complexes (also known as lipoplexes) are desirable non-viral gene vectors because of low immunogenicity, ease of scale-up procedures, etc.. To be viable for in vivo applications, CL-DNA complexes need to be stable in circulation, which can be achieved by addition of poly-(ethylene glycol) (PEG)-lipids. Unfortunately, PEG-lipids interfere with complex-cell membrane interactions that are vital for particle attachment and uptake and can inhibit membrane fusion required for endosomal escape. The benefit of increased stability from PEG-lipids, thus, comes at the cost of a reduction in transfection efficiency (TE).To recover the resulting loss in TE, we have designed and synthesized a hydrolysable acid-labile PEG-lipid (HPEG-lipid, PEG MW 2000) which is stable at physiological pH, but is cleaved at low pH. The natural acidification process of endosomes will liberate the PEG from the lipoplexes. We have studied the colloidal stability and transfection efficiency of these HPEG-lipoplexes in mammalian cell cultures. The acid sensitivity and the colloidal stability were characterized by TLC and dynamic light scattering, respectively. The HPEG-lipid is stable at neutral pH for more than 24 h, but degrades completely within 1 h at pH 4, leading to particle aggregation. HPEG-lipoplexes show lower toxicity and enhanced TE in comparison to lipoplexes stabilized with pH-stable PEG-lipids. Live-cell images show that both pH-sensitive and pH-stable PEG-lipoplexes were internalized to quantitatively similar particle distributions within the first 2 h of incubation. Thus, the increased TE of the HPEG-lipoplexes can be attributed to efficient endosomal escape, enabled by the novel HPEG-lipid. Funded by NIH-GM-59288 and NSF-DMR-1101900.[1] Ewert, K. K. et al, Topics Curr. Chem. 2010, 296, 191-226

Computational study of bindings of HK20 Fab and D5 Fab to HIV-1 gp41

Antibodies HK20 and D5 have been shown to target HIV-1 gp41, thereby inhibiting membrane fusion that facilitates viral entry. The binding picture is static, based on the X-ray crystal structures of the Fab regions and gp41 mimetic five-helix bundle. In this study, we carried out molecular dynamics simulation to provide the dynamic binding picture. Calculated binding free energies are within reasonable range of and follow the trend of the experimental values: −15.28kcal/mol for HK20 Fab (expt. −11.60kcal/mol) and −17.90kcal/mol for D5 Fab (expt. −11.70kcal/mol). Alanine scanning at protein–protein interface reveals that the highest contributors to binding for HK20 Fab are F54 and I56, both of VH region, as well as R30′ of VL region; whereas for D5 Fab, F54 of VH region, as well as W32′ and Y94′ of VL region. HK20 F54 and I56, as well as D5 I52, F54, and T56, bind to the gp41 hydrophobic binding pocket, an important region targeted by many other fusion inhibitors. Hydrogen bonding analysis also identifies high-occupancy hydrogen bonds at the periphery of gp41 hydrophobic pocket. Considering that almost all interface residues are turn residues, further work may be directed to turn mimics. Pre-orientation by the hydrogen bonds to poise this particular turn towards the binding pocket may also be a point worth pursuing.

Capturing a Fusion Intermediate of Influenza Hemagglutinin with a Cholesterol-conjugated Peptide, a New Antiviral Strategy for Influenza Virus

We previously described fusion-inhibitory peptides that are targeted to the cell membrane by cholesterol conjugation and potently inhibit enveloped viruses that fuse at the cell surface, including HIV, parainfluenza, and henipaviruses. However, for viruses that fuse inside of intracellular compartments, fusion-inhibitory peptides have exhibited very low antiviral activity. We propose that for these viruses, too, membrane targeting via cholesterol conjugation may yield potent compounds. Here we compare the activity of fusion-inhibitory peptides derived from the influenza hemagglutinin (HA) and show that although the unconjugated peptides are inactive, the cholesterol-conjugated compounds are effective inhibitors of infectivity and membrane fusion. We hypothesize that the cholesterol moiety, by localizing the peptides to the target cell membrane, allows the peptides to follow the virus to the intracellular site of fusion. The cholesterol-conjugated peptides trap HA in a transient intermediate state after fusion is triggered but before completion of the refolding steps that drive the merging of the viral and cellular membranes. These results provide proof of concept for an antiviral strategy that is applicable to intracellularly fusing viruses, including known and emerging viral pathogens.

Fusion failure of dense-cored proacrosomal vesicles in an inducible mouse model of male infertility

The acrosome is a specialized secretory vesicle located in the head of spermatozoa and has an essential role during fertilization. This organelle and the sperm nucleus have aberrant morphologies in forms of male infertility in humans (teratozoospermia), often associated with poor motility (asthenoteratozoospermia). To further our understanding of the aetiology of these conditions, we have performed a pathological investigation of a model of asthenoteratozoospermia that can be induced in mice by N-butyldeoxynojirimycin (NB-DNJ). We have found that, in mice treated with NB-DNJ, instead of an acrosome forming over the round spermatid nucleus, multivesicular bodies (MVB) accumulate in the vicinity of this nucleus. Electron microscopy has revealed that proacrosomic vesicles or granules (PAG) secreted during the Golgi phase of spermiogenesis do not fuse together to form an acrosomic vesicle, but rather attach transiently to the spermatid nucleus. Immunocytochemistry has shown that acrosomal membrane proteins and cytosolic acrosome-associated proteins are redirected to MVB in affected testes, whereas glycoproteins originating in the dense core of the PAG are degraded. Thus, the major effect of NB-DNJ is to inhibit membrane fusion of Golgi-derived secretory vesicles destined for acrosome formation, raising the possibility that these vesicles are critically affected in forms of (astheno)teratozoospermia.

Biophysical Investigations of GBV‐C E1 Peptides as Potential Inhibitors of HIV‐1 Fusion Peptide

Five peptide sequences corresponding to the E1 protein of GBV-C [NCCAPEDIGFCLEGGCLV (P7), APEDIGFCLEGGCLVALG (P8), FCLEGGCLVALGCTICTD (P10), QAGLAVRPGKSAAQLVGE (P18), and AQLVGELGSLYGPLSVSA (P22)] were synthesized because they were capable of interfering with the HIV-1 fusion peptide (HIV-1 FP)-vesicle interaction. In this work the interaction of these peptides with the HIV-1 FP, as well as with membrane models, was analyzed to corroborate their inhibition ability and to understand if the interaction with the fusion peptide takes place in solution or at the membrane level. Several studies were carried out on aggregation and membrane fusion, surface Plasmon resonance, and conformational analysis by circular dichroism. Moreover, in vitro toxicity assays, including cytotoxicity studies in 3T3 fibroblasts and hemolysis assays in human red blood cells, were performed to evaluate if these peptides could be potentially used in anti-HIV-1 therapy. Results show that P10 is not capable of inhibiting membrane fusion caused by HIV-1 and it aggregates liposomes and fuses membranes, thus we decided to discard it for futures studies. P18 and P22 do not inhibit membrane fusion, but they inhibit the ability of HIV-1 FP to form pores in bilayers, thus we have not discarded them yet. P7 and P8 were selected as the best candidates for future studies because they are capable of inhibiting membrane fusion and the interaction of HIV-1 FP with bilayers. Therefore, these peptides could be potentially used in future anti-HIV-1 research.

Resolving the Function of Distinct Munc18-1/SNARE Protein Interaction Modes in a Reconstituted Membrane Fusion Assay

Sec1p/Munc18 proteins and SNAP receptors (SNAREs) are key components of the intracellular membrane fusion machinery. Compartment-specific v-SNAREs on a transport vesicle pair with their cognate t-SNAREs on the target membrane and drive lipid bilayer fusion. In a reconstituted assay that dissects the sequential assembly of t-SNARE (syntaxin 1·SNAP-25) and v-/t-SNARE (VAMP2·syntaxin 1·SNAP-25) complexes, and finally measures lipid bilayer merger, we resolved the inhibitory and stimulatory functions of the Sec1p/Munc18 protein Munc18-1 at the molecular level. Inhibition of membrane fusion by Munc18-1 requires a closed conformation of syntaxin 1. Remarkably, the concurrent preincubation of Munc18-1-inhibited syntaxin 1 liposomes with both VAMP2 liposomes and SNAP-25 at low temperature releases the inhibition and effectively stimulates membrane fusion. VAMP8 liposomes can neither release the inhibition nor exert the stimulatory effect, demonstrating the need for a specific Munc18-1/VAMP2 interaction. In addition, Munc18-1 binds to the N-terminal peptide of syntaxin 1, which is obligatory for a robust stimulation of membrane fusion. In contrast, this interaction is neither required for the inhibitory function of Munc18-1 nor for the release of this block. These results indicate that Munc18-1 and the neuronal SNAREs already have the inherent capability to function as a basic stage-specific off/on switch to control membrane fusion.

Complexin activates and clamps SNAREpins by a common mechanism involving an intermediate energetic state

The core mechanism of intracellular vesicle fusion consists of SNAREpin zippering between vesicular and target membranes. Recent studies indicate that the same SNARE-binding protein, complexin (CPX), can act either as a facilitator or as an inhibitor of membrane fusion, constituting a controversial dilemma. Here we take energetic measurements with the surface force apparatus that reveal that CPX acts sequentially on assembling SNAREpins, first facilitating zippering by nearly doubling the distance at which v- and t-SNAREs can engage and then clamping them into a half-zippered fusion-incompetent state. Specifically, we find that the central helix of CPX allows SNAREs to form this intermediate energetic state at 9-15 nm but not when the bilayers are closer than 9 nm. Stabilizing the activated-clamped state at separations of less than 9 nm requires the accessory helix of CPX, which prevents membrane-proximal assembly of SNAREpins.

Effects of synaptotagmin 2 on membrane fusion between liposomes that contain SNAREs involved in exocytosis in mast cells

Mast cells play a pivotal role in allergic responses. Antigen stimulation causes elevation of the intracellular Ca2+ concentration, which triggers the exocytotic release of inflammatory mediators such as histamine. Recent research, including our own, has revealed that SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins such as syntaxin-3, -4, SNAP-23, and VAMP-8 are involved in exocytosis. Although exocytosis in mast cells is Ca2+ dependent, the target molecule that interacts with Ca2+ is not clear. Synaptotagmin is a Ca2+ sensor and regulates exocytosis in neuronal cells. However, the role of synaptotagmin 2, a member of the synaptotagmin family, in exocytosis in mast cells remains controversial. In this study, we investigated the role of synaptotagmin 2 by a liposome-based fusion assay. SNARE proteins (SNAP-23, syntaxin-3, VAMP-8) and synaptotagmin 2 were expressed in Escherichia coli and purified as GST-tagged or His-tagged fusion proteins. These SNARE proteins were incorporated by a detergent dialysis method. Membrane fusion between liposomes was monitored by fluorescence resonance energy transfer between fluorescent-labeled phospholipids. In the presence of Ca2+, low synaptotagmin 2 concentration inhibited membrane fusion between SNARE-containing liposomes, while high synaptotagmin 2 concentration enhanced membrane fusion. This enhancement required phosphatidylserine as a membrane component. These results suggest that synaptotagmin 2 regulates membrane fusion of SNARE-containing liposomes involved in exocytosis in mast cells, and that this regulation is dependent on synaptotagmin 2 concentration, Ca2+, and phosphatidylserine.

Membrane fusion by the GTPase atlastin requires a conserved C-terminal cytoplasmic tail and dimerization through the middle domain

The biogenesis and maintenance of the endoplasmic reticulum (ER) requires membrane fusion. ER homotypic fusion is driven by the large GTPase atlastin. Domain analysis of atlastin shows that a conserved region of the C-terminal cytoplasmic tail is absolutely required for fusion activity. Atlastin in adjacent membranes must associate to bring the ER membranes into molecular contact. Drosophila atlastin dimerizes in the presence of GTPγS but is monomeric with GDP or without nucleotide. Oligomerization requires the juxtamembrane middle domain three-helix bundle, as does efficient GTPase activity. A soluble version of the N-terminal cytoplasmic domain that contains the GTPase domain and the middle domain three-helix bundle serves as a potent, concentration-dependent inhibitor of membrane fusion both in vitro and in vivo. However, atlastin domains lacking the middle domain are without effect. GTP-dependent dimerization of atlastin generates an enzymatically active protein that drives membrane fusion after nucleotide hydrolysis and conformational reorganization.

A Computational Approach to Search Active Peptides as Membrane Fusion Inhibitors of HIV-1

A Computational Approach to Search Active Peptides as Membrane Fusion Inhibitors of HIV-1

Functional analysis of RhoGDI inhibitory activity on vacuole membrane fusion

RhoGDIs (Rho GDP-dissociation inhibitors) are the natural inhibitors of Rho GTPases. They interfere with Rho protein function by either blocking upstream activation or association with downstream signalling molecules. RhoGDIs can also extract membrane-bound Rho GTPases to form soluble cytosolic complexes. We have shown previously that purified yeast RhoGDI Rdi1p, can inhibit vacuole membrane fusion in vitro. In the present paper we functionally dissect Rdi1p to discover its mode of regulating membrane fusion. Overexpression of Rdi1p in vivo profoundly affected cell morphology including increased actin patches in mother cells indicative of polarity defects, delayed ALP (alkaline phosphatase) sorting and the presence of highly fragmented vacuoles indicative of membrane fusion defects. These defects were not caused by the loss of typical transport and fusion proteins, but rather were linked to the reduction of membrane localization and activation of Cdc42p and Rho1p. Subcellular fractionation showed that Rdi1p is predominantly a cytosolic monomer, free of bound Rho GTPases. Overexpression of endogenous Rdi1p, or the addition of exogenous Rdi1p, generated stable cytosolic complexes. Rdi1p structure-function analysis showed that membrane association via the C-terminal β-sheet domain was required for the functional inhibition of membrane fusion. Furthermore, Rdi1p inhibited membrane fusion through the binding of Rho GTPases independent from its extraction activity.

Membrane topology analysis of HIV-1 envelope glycoprotein gp41

BackgroundThe gp41 subunit of the HIV-1 envelope glycoprotein (Env) has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD). An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP) that is only active in the cytoplasm. The tag protein (HaloTag) and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system.ResultsIn the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells) supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells), the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability.ConclusionsIt is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD.

Inner/Outer Nuclear Membrane Fusion in Nuclear Pore Assembly

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.