Search

Oncolytic viruses are promising cancer therapies due to their selective killing of tumor cells and ability to stimulate the host immune system. As an oncolytic virus platform, vaccinia virus has unique advantages, including rapid replication, a broad range of host targets, and a large capacity for transgene incorporation. In this study, we developed a novel oncolytic vaccinia virus with high potency and a favorable safety profile. We began with the International Health Department-White (IHD-W) strain, which had the strongest cytotoxicity against tumor cells among the four vaccinia virus strains tested. Next, several candidate viruses were constructed by deleting three viral genes (C11R, K3L, and J2R) in various combinations, and their efficacy and safety were compared. The virus ultimately selected, named KLS-3010, exhibited strong antitumor activity against broad targets in vitro and in vivo. Furthermore, KLS-3010 showed a favorable safety profile in mice, as determined by the biodistribution and body weight change. More promisingly, KLS-3010 was able to shift the tumor microenvironment to a proinflammatory state, as evidenced by an increase in activated lymphocytes after KLS-3010 administration, suggesting that this strain may elicit an oncolytic virus-mediated immune response. The KLS-3010 strain thus represents a promising platform for the further development of oncolytic virus-based cancer therapies.

Neurons have the remarkable ability to release a batch of neurotransmitters into the synapse immediately after an action potential. This signature event is made possible by the simultaneous fusion of a number of synaptic vesicles to the plasma membrane upon Ca2+ entry into the active zone. The outcomes of both cellular and in vitro studies suggest that soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) and synaptotagmin 1 (Syt1) constitute the minimal fast exocytosis machinery in the neuron. Syt1 is the major Ca2+-sensor and orchestrates the synchronous start of individual vesicle fusion events while SNAREs are the membrane fusion machinery that dictates the kinetics of each single fusion event. The data also suggest that Ca2+-bound Syt1 is involved in the upstream docking step which leads to an increase in the number of fusion events or the size of the release, leaving the SNARE complex alone to carry out membrane fusion by themselves.

Membrane scaffold protein (MSP) is a versatile protein that can be used to study diverse membrane proteins. MSP is strongly expressed in E. coli; however, applications of MSP in in vivo studies remain limited because of contamination with large amounts of endotoxins. Endotoxins cannot be easily removed from MSP following standard purification protocols for His6-tagged proteins, washing with detergents, or Q-Sepharose anion exchange chromatography, regardless of whether the expression host is E. coli BL21(DE3) or ClearColi BL21(DE3). Furthermore, the concentrations of MSP-bound endotoxins were not reduced during nanodisc formation, such that the assembled nanodiscs still contained significant amounts of endotoxins. We hypothesized that the structural properties of MSP that are responsible for membrane scaffolding mediated the strong binding between MSP and the endotoxins. We showed that partial denaturation of MSP with 2 M urea effectively disrupted MSP-endotoxin interactions. MSP-bound endotoxins were successfully removed via Q-Sepharose chromatography following urea treatment. The combined treatment with urea and Q-Sepharose resulted in ∼80-fold reduction in the specific endotoxin level relative to that of conventional Ni-NTA chromatography combined with detergent treatment. The low endotoxin level of 2.0 EU/mg MSP obtained in this study makes it suitable for applications in animal studies.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion by forming a ternary SNARE complex. A minimalist approach utilizing proteoliposomes with reconstituted SNARE proteins yielded a wealth of information pinpointing the molecular mechanism of SNARE-mediated fusion and its regulation by accessory proteins. Two important attributes of a membrane fusion are lipid-mixing and the formation of an aqueous passage between apposing membranes. These two attributes are typically observed by using various fluorescent dyes. Currently available in vitro assay systems for observing fusion pore opening have several weaknesses such as cargo-bleeding, incomplete removal of unencapsulated dyes, and inadequate information regarding the size of the fusion pore, limiting measurements of the final stage of membrane fusion. In the present study, we used a biotinylated green fluorescence protein and streptavidin conjugated with Dylight 594 (DyStrp) as a Föster resonance energy transfer (FRET) donor and acceptor, respectively. This FRET pair encapsulated in each v-vesicle containing synaptobrevin and t-vesicle containing a binary acceptor complex of syntaxin 1a and synaptosomal-associated protein 25 revealed the opening of a large fusion pore of more than 5 nm, without the unwanted signals from unencapsulated dyes or leakage. This system enabled determination of the stoichiometry of the merging vesicles because the FRET efficiency of the FRET pair depended on the molar ratio between dyes. Here, we report a robust and informative assay for SNARE-mediated fusion pore opening.

Dynamic light scattering (DLS) spectroscopy provides rapid information on the size distribution of a large number of particles in a mixture. Vesicle sizes change during the merger of lipid bilayers, and DLS analysis can provide rapid, accurate, and non-perturbative quantification of the size distribution of proteoliposomes in SNARE-dependent membrane fusion. In this chapter, we describe the methodologies and reagents used for DLS spectroscopy in a biochemical and biophysical study of SNARE-mediated membrane fusion.

Dynamic light scattering (DLS) spectroscopy provides rapid information on the size distribution of a large number of particles in a mixture. Vesicle sizes change during the merger of lipid bilayers, and DLS analysis can provide rapid, accurate, and non-perturbative quantification of the size distribution of proteoliposomes in SNARE-dependent membrane fusion. In this chapter, we describe the methodologies and reagents used for DLS spectroscopy in a biochemical and biophysical study of SNARE-mediated membrane fusion.

Neurons have the remarkable ability to release a batch of neurotransmitters into the synapse immediately after an action potential. This signature event is made possible by the simultaneous fusion of a number of synaptic vesicles to the plasma membrane upon Ca(2+) entry into the active zone. The outcomes of both cellular and in vitro studies suggest that soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) and synaptotagmin 1 (Syt1) constitute the minimal fast exocytosis machinery in the neuron. Syt1 is the major Ca(2+)-sensor and orchestrates the synchronous start of individual vesicle fusion events while SNAREs are the membrane fusion machinery that dictates the kinetics of each single fusion event. The data also suggest that Ca(2+)-bound Syt1 is involved in the upstream docking step which leads to an increase in the number of fusion events or the size of the release, leaving the SNARE complex alone to carry out membrane fusion by themselves.

Vesicle-associated V-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and target membrane-associated T-SNAREs (syntaxin 4 and SNAP-23) assemble into a core trans-SNARE complex that mediates membrane fusion during mast cell degranulation. This complex plays pivotal roles at various stages of exocytosis from the initial priming step to fusion pore opening and expansion, finally resulting in the release of the vesicle contents. In this study, peptides with the sequences of various SNARE motifs were investigated for their potential inhibitory effects against SNARE complex formation and mast cell degranulation. The peptides with the sequences of the N-terminal regions of vesicle-associated membrane protein 2 (VAMP2) and VAMP8 were found to reduce mast cell degranulation by inhibiting SNARE complex formation. The fusion of protein transduction domains to the N-terminal of each peptide enabled the internalization of the fusion peptides into the cells equally as efficiently as cell permeabilization by streptolysin-O without any loss of their inhibitory activities. Distinct subsets of mast cell granules could be selectively regulated by the N-terminal-mimicking peptides derived from VAMP2 and VAMP8, and they effectively decreased the symptoms of atopic dermatitis in mouse models. These results suggest that the cell membrane fusion machinery may represent a therapeutic target for atopic dermatitis.

Soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins generate energy required for membrane fusion. They form a parallelly aligned four-helix bundle called the SNARE complex, whose formation is initiated from the N terminus and proceeds toward the membrane-proximal C terminus. Previously, we have shown that this zippering-like process can be controlled by several flavonoids that bind to the intermediate structures formed during the SNARE zippering. Here, our aim was to test whether the fluorescence resonance energy transfer signals that are observed during the inner leaflet mixing assay indeed represent the hemifused vesicles. We show that changes in vesicle size accompanying the merging of bilayers is a good measure of progression of the membrane fusion. Two merging vesicles with the same size D in diameter exhibited their hydrodynamic diameters 2D + d (d, intermembrane distance), 2D and 2D as membrane fusion progressed from vesicle docking to hemifusion and full fusion, respectively. A dynamic light scattering assay of membrane fusion suggested that myricetin stopped membrane fusion at the hemifusion state, whereas delphinidin and cyanidin prevented the docking of the vesicles. These results are consistent with our previous findings in fluorescence resonance energy transfer assays.

Epothilone B (EpoB) is a paclitaxel (PTX)-like microtubule-stabilizing agent that induces mitotic arrest and apoptosis in cells. EpoB is a promising anti-tumor agent, and is thought to have the potential to overcome well-known PTX resistance. However, EpoB is barely soluble in water, and is fatal to normal cells due to extremely potent cytotoxicity. To reduce the unwanted cytotoxicity of EpoB to normal cells, a reconstituted highdensity lipoprotein (rHDL)-like nanoparticle of EpoB (EpoB-rHDL) was assembled using apolipoprotein A-I (apoA-I). The EpoB-rHDL, as well as PTX-rHDL (a HDLlike nanoparticle of paclitaxel), were indeed mild (nontoxic) to certain cell lines such as MCF7, MDA-MB-231, and SK-OV-3, while free EpoB and PTX were very toxic to those same cells. In contrast, the EpoB-rHDL and PTXrHDL were very effective in killing the Caco-2 and ZR-75-1 while free drugs were less toxic to those cells. The susceptibility of cell lines to rHDLs was dependent on the expression of scavenger receptor class B type I (SR-BI), indicating that EpoB-rHDL selectively and efficiently kills only SR-BI-overexpressing cells. Furthermore, the EpoBrHDL released EpoB only at acidic pH, which may facilitate the escape of drugs from acidic endosome. Thus, EpoB-rHDL shown in this study enables safe and targeted delivery of the potent EpoB to cancer cells in SR-BIdependent manner.

Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate (5-IP7) are highly energetic inositol metabolites containing phosphoanhydride bonds. Although inositol pyrophosphates are known to regulate various biological events, including growth, survival, and metabolism, the molecular sites of 5-IP7 action in vesicle trafficking have remained largely elusive. We report here that elevated 5-IP7 levels, caused by overexpression of inositol hexakisphosphate (IP6) kinase 1 (IP6K1), suppressed depolarization-induced neurotransmitter release from PC12 cells. Conversely, IP6K1 depletion decreased intracellular 5-IP7 concentrations, leading to increased neurotransmitter release. Consistently, knockdown of IP6K1 in cultured hippocampal neurons augmented action potential-driven synaptic vesicle exocytosis at synapses. Using a FRET-based in vitro vesicle fusion assay, we found that 5-IP7, but not 1-IP7, exhibited significantly higher inhibitory activity toward synaptic vesicle exocytosis than IP6 Synaptotagmin 1 (Syt1), a Ca(2+) sensor essential for synaptic membrane fusion, was identified as a molecular target of 5-IP7 Notably, 5-IP7 showed a 45-fold higher binding affinity for Syt1 compared with IP6 In addition, 5-IP7-dependent inhibition of synaptic vesicle fusion was abolished by increasing Ca(2+) levels. Thus, 5-IP7 appears to act through Syt1 binding to interfere with the fusogenic activity of Ca(2+) These findings reveal a role of 5-IP7 as a potent inhibitor of Syt1 in controlling the synaptic exocytotic pathway and expand our understanding of the signaling mechanisms of inositol pyrophosphates.

Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca(2+) stimulates fusion-pore formation in a millisecond time scale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca(2+). When 10 μM Ca(2+) was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery composed of SNAREs and synaptotagmin 1 is capable of driving membrane fusion in a millisecond time scale when a proper vesicle priming is established. The chemical controller of SNARE-driven membrane fusion should serve as a versatile tool for investigating the differential roles of various synaptic proteins in discrete fusion steps.

Fusion of synaptic vesicles with the presynaptic plasma membrane in the neuron is mediated by soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor (SNARE) proteins. SNARE complex formation is a zippering-like process which initiates at the N-terminus and proceeds to the C-terminal membrane-proximal region. Previously, we showed that this zippering-like process is regulated by several polyphenols, leading to the arrest of membrane fusion and the inhibition of neuroexocytosis. In vitro studies using purified SNARE proteins reconstituted in liposomes revealed that each polyphenol uniquely regulates SNARE zippering. However, the unique regulatory effect of each polyphenol in cells has not yet been examined. In the present study, we observed SNARE zippering in neuronal PC12 cells by measuring the fluorescence resonance energy transfer (FRET) changes of a cyan fluorescence protein (CFP) and a yellow fluorescence protein (YFP) fused to the N-termini or C-termini of SNARE proteins. We show that delphinidin and cyanidin inhibit the initial N-terminal nucleation of SNARE complex formation in a Ca(2+)-independent manner, while myricetin inhibits Ca(2+)-dependent transmembrane domain association of the SNARE complex in the cell. This result explains how polyphenols exhibit botulinum neurotoxin-like activity in vivo.

Anti-allergic effects of dietary polyphenols were extensively studied in numerous allergic disease models, but the molecular mechanisms of anti-allergic effects by polyphenols remain poorly understood. In the present study, we show that the release of granular cargo molecules, contained in distinct subsets of granules of mast cells, is specifically mediated by two sets of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, and that various polyphenols differentially inhibit the formation of those SNARE complexes. Expression analysis of RBL-2H3 cells for 11 SNARE genes and a lipid mixing assay of 24 possible combinations of reconstituted SNAREs indicated that the only two active SNARE complexes involved in mast cell degranulation are Syn (syntaxin) 4/SNAP (23 kDa synaptosome-associated protein)-23/VAMP (vesicle-associated membrane protein) 2 and Syn4/SNAP-23/VAMP8. Various polyphenols selectively or commonly interfered with ternary complex formation of these two SNARE complexes, thereby stopping membrane fusion between granules and plasma membrane. This led to the differential effect of polyphenols on degranulation of three distinct subsets of granules. These results suggest the possibility that formation of a variety of SNARE complexes in numerous cell types is controlled by polyphenols which, in turn, might regulate corresponding membrane trafficking.

Jae-Yoon Shin,1,* Yoosoo Yang,1,* Paul Heo,1 Ji-Chun Lee,1 ByoungJae Kong,1 Jae Youl Cho,1 Keejung Yoon,1 Cheol-Su Shin,2 Jin-Ho Seo,3 Sung-Gun Kim,4 Dae-Hyuk Kweon11Department of Genetic Engineering, College of Biotechnology and Bioengineering, and Center for Human Interface Nano Technology, Sungkyunkwan University, 2APTech Research Center, Suwon, 3Department of Agricultural Biotechnology, Seoul National University, Seoul, 4Department of Biomedical Science, Youngdong University, Chungbuk, South Korea*These authors contributed equally to this workBackground: Nanoparticles undergoing physicochemical changes to release enclosed drugs at acidic pH conditions are promising vehicles for antitumor drug delivery. Among the various drug carriers, high-density lipoprotein (HDL)-like nanoparticles have been shown to be beneficial for cancer chemotherapy, but have not yet been designed to be pH-responsive.Methods and results: In this study, we developed a pH-responsive HDL-like nanoparticle that selectively releases paclitaxel, a model antitumor drug, at acidic pH. While the well known HDL-like nanoparticle containing phospholipids, phosphatidylcholine, and apolipoprotein A-I, as well as paclitaxel (PTX-PL-NP) was structurally robust at a wide range of pH values (3.8–10.0), the paclitaxel nanoparticle that only contained paclitaxel and apoA-I selectively released paclitaxel into the medium at low pH. The paclitaxel nanoparticle was stable at physiological and basic pH values, and over a wide range of temperatures, which is a required feature for efficient cancer chemotherapy. The homogeneous assembly enabled high paclitaxel loading per nanoparticle, which was 62.2% (w/w). The molar ratio of apolipoprotein A-I and paclitaxel was 1:55, suggesting that a single nanoparticle contained approximately 110 paclitaxel particles in a spherical structure with a 9.2 nm diameter. Among the several reconstitution methods applied, simple dilution following sonication enhanced the reconstitution yield of soluble paclitaxel nanoparticles, which was 0.66. As a result of the pH responsiveness, the anticancer effect of paclitaxel nanoparticles was much more potent than free paclitaxel or PTX-PL-NP.Conclusion: The anticancer efficacy of both paclitaxel nanoparticles and PTX-PL-NP was dependent on the expression of scavenger receptor class B type I, while the killing efficacy of free paclitaxel was independent of this receptor. We speculate that the pH responsiveness of paclitaxel nanoparticles enabled efficient endosomal escape of paclitaxel before lysosomal break down. This is the first report on pH-responsive nanoparticles that do not contain any synthetic polymer.Keywords: pH responsiveness, nanoparticle, apolipoprotein A-I, paclitaxel

Inositol pyrophosphates such as IP7 (5-diphosphoinositol pentakisphosphate) are highly energetic inositol metabolites containing pyrophosphate bonds. While IP7 are known to regulate various biological events like metabolism, the molecular site of IP7 action in vesicle trafficking remains elusive. Here, we showed that IP7 potently inhibits synaptic vesicle exocytosis in both invitro reconstitution and cell culture settings. IP7 was identified as a high-affinity ligand for synaptotagmin 1 (Syt1), a calcium sensor essential for membrane fusion. Interactions of IP7 with Syt1 reduced the binding between Syt1 and Ca2+ as well as endowed Syt1 with its own negative effect, thereby suppressing Syt1 activation. These findings reveal a role of IP7 as an inhibitor of the exocytotic pathway and expand our understanding of the signaling mechanisms of inositol pyrophosphates.