Byoungjae Kong

pH-responsive high-density lipoprotein-like nanoparticles to release paclitaxel at acidic pH in cancer chemotherapy

Background 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.

Polyphenols differentially inhibit degranulation of distinct subsets of vesicles in mast cells by specific interaction with granule-type-dependent SNARE complexes

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.

Dynamic light scattering analysis of SNARE-driven membrane fusion and the effects of SNARE-binding flavonoids

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.

Hemifusion in Synaptic Vesicle Cycle

In the neuron, early neurotransmitters are released through the fusion pore prior to the complete vesicle fusion. It has been thought that the fusion pore is a gap junction-like structure made of transmembrane domains (TMDs) of soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins. However, evidence has accumulated that lipid mixing occurs prior to the neurotransmitter release through the fusion pore lined predominantly with lipids. To explain these observations, the hemifusion, a membrane structure in which two bilayers are partially merged, has emerged as a key step preceding the formation of the fusion pore. Furthermore, the hemifusion appears to be the bona fide intermediate step not only for the synaptic vesicle cycle, but for a wide range of membrane remodeling processes, such as viral membrane fusion and endocytotic membrane fission.

Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis

Significance Inositol phosphates have long been considered to be negative regulators of synaptic exocytosis, but the function of diphosphoinositol pentakisphosphate (IP7) has remained elusive. We found that overexpression and depletion of inositol hexakisphosphate (IP6) kinase in PC12 cells or hippocampal neurons led to a reduction and increase in neurotransmitter release, respectively. Biophysical assays revealed that 5-IP7 inhibited Ca2+-induced synaptic membrane fusion at a concentration one order of magnitude lower than that required for IP6. We further elucidated the molecular mechanism responsible for 5-IP7 actions, demonstrating that 5-IP7 directly bound with high affinity to synaptotagmin 1 (Syt1), a Ca2+ sensor in cellular exocytosis, and suppressed its fusogenic activity. Thus, our data propose 5-IP7 as a potent inhibitor of Syt1 actions on Ca2+-mediated synaptic vesicle fusion. 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 Ca2+ 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 Ca2+ levels. Thus, 5-IP7 appears to act through Syt1 binding to interfere with the fusogenic activity of Ca2+. 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.

A Chemical Controller of SNARE-Driven Membrane Fusion That Primes Vesicles for Ca2+-Triggered Millisecond Exocytosis

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.

Dynamic Light Scattering Analysis to Dissect Intermediates 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.

Search for a minimal machinery for Ca 2+ -triggered millisecond neuroexocytosis

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.

Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor-Derived Peptides for Regulation of Mast Cell Degranulation

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.

SNARE zippering is hindered by polyphenols in the neuron

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.